Superoxide dismutase 1 expression is modulated by the core pluripotency transcription factors Oct4, Sox2 and Nanog in embryonic stem cells.

PubMed

Solari, Claudia; Petrone, María Victoria; Echegaray, Camila Vázquez; Cosentino, María Soledad; Waisman, Ariel; Francia, Marcos; Barañao, Lino; Miriuka, Santiago; Guberman, Alejandra

2018-06-19

Redox homeostasis is vital for cellular functions and to prevent the detrimental consequences of oxidative stress. Pluripotent stem cells (PSCs) have an enhanced antioxidant system which supports the preservation of their genome. Besides, reactive oxygen species (ROS) are proposed to be involved in both self-renewal maintenance and in differentiation in embryonic stem cells (ESCs). Increasing evidence shows that cellular systems related to the oxidative stress defense decline along differentiation of PSCs. Although redox homeostasis has been extensively studied for many years, the knowledge about the transcriptional regulation of the genes involved in these systems is still limited. In this work, we studied Sod1 gene modulation by the PSCs fundamental transcription factors Oct4, Sox2 and Nanog. We found that this gene, which is expressed in mouse ESCs (mESCs), was repressed when they were induced to differentiate. Accordingly, these factors induced Sod1 promoter activity in a trans-activation assay. Finally, Sod1 mRNA levels were reduced when Oct4, Sox2 and Nanog were down-regulated by a shRNA approach in mESCs. Taken together, we found that PSCs' key transcription factors are involved in the modulation of Sod1 gene, suggesting a relationship between the pluripotency core and redox homeostasis in these cells. Copyright © 2018. Published by Elsevier B.V.

CRISP-3, a protein with homology to plant defense proteins, is expressed in mouse B cells under the control of Oct2.

PubMed

Pfisterer, P; König, H; Hess, J; Lipowsky, G; Haendler, B; Schleuning, W D; Wirth, T

1996-11-01

The Oct2 transcription factor is expressed throughout the B-lymphoid lineage and plays an essential role during the terminal phase of B-cell differentiation. Several genes specifically expressed in B lymphocytes have been identified that contain a functional octamer motif in their regulatory elements. However, expression of only a single gene, the murine CD36 gene, has been shown to date to be dependent on Oct2. Here, we present the identification and characterization of a further gene, coding for cysteine-rich secreted protein 3 (CRISP-3), whose expression in B cells is regulated by Oct2. We show that CRISP-3 is expressed in the B-lymphoid lineage specifically at the pre-B-cell stage. By using different experimental strategies, including nuclear run-on experiments, we demonstrate that this gene is transcriptionally activated by Oct2. Furthermore, analysis of CRISP-3 expression in primary B cells derived from either wild-type or Oct2-deficient mice demonstrates the dependence on Oct2. Two variant octamer motifs were identified in the upstream promoter region of the crisp-3 gene, and Oct2 interacts with both of them in vitro. Cotransfection experiments with expression vectors for Oct1 and Oct2 together with a reporter driven by the crisp-3 promoter showed that transcriptional activation of this promoter can only be achieved with Oct2. The C-terminal transactivation domain of Oct2 is required for this activation. Finally, introducing specific mutations in the two variant octamer motifs revealed that both of them are important for full transcriptional activation by Oct2.

Targeting Tumor Oct4 to Deplete Prostate Tumor and Metastasis Initiating Cells

DTIC Science & Technology

2017-12-01

Nie, POU5F1B, an OCT4 Retrogene, Suppresses Uncontrolled Tumor Growth. Keystone Meeting on Molecular and Cellular Basis of Growth and Regeneration...Daotai Nie. Cancer Stem Cells in Resistance to Cytotoxic Drugs: Implications in Chemotherapy. B. Bonavida (ed.), Molecular Mechanisms of Tumor Cell...retrogene of the master embryonic stem cell gene POU5F1 is associated with prostate cancer susceptibility. American journal of human genetics 94

Targeting Tumor Oct4 to Deplete Prostate Tumor and Metastasis Initiating Cells

DTIC Science & Technology

2016-10-01

Award Number: W81XWH-13-1-0461 TITLE: Targeting Tumor Oct4 to Deplete Prostate Tumor- and Metastasis-Initiating Cells PRINCIPAL INVESTIGATOR: Daotai...29 2016 4. TITLE AND SUBTILE Targeting Tumor Oct4 to Deplete Prostate Tumor- and Metastasis-Initiating Cells 5a. CONTRACT NUMBER 5b. GRANT NUMBER...the c-MYC oncogene. POU5F1B is a pseudogene of embryonic Oct4 (POU5F1). A recent study found that tumor Oct4 found in prostate cancer cells is due

Targeting Tumor Oct4 to Deplete Prostate Tumor- and Metastasis-Initiating Cells

DTIC Science & Technology

2015-10-01

and stem cell To investigate whether POU5F1B overrxpression can induce cancer stem cell -related genes expression, we did cancer stem cell ...future 15. SUBJECT TERMS OCT4, cancer stem cells , prostate cancer, metastasis, tumor formation 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT...described in last report. Here we describe some findings previously not reported. 1.1 POU5F1B expression in prostatic tissue As cancer stem cell marker

Genome editing reveals a role for OCT4 in human embryogenesis.

PubMed

Fogarty, Norah M E; McCarthy, Afshan; Snijders, Kirsten E; Powell, Benjamin E; Kubikova, Nada; Blakeley, Paul; Lea, Rebecca; Elder, Kay; Wamaitha, Sissy E; Kim, Daesik; Maciulyte, Valdone; Kleinjung, Jens; Kim, Jin-Soo; Wells, Dagan; Vallier, Ludovic; Bertero, Alessandro; Turner, James M A; Niakan, Kathy K

2017-10-05

Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.

[Functional analysis of Oct4 promoter in Xuhuai goat].

PubMed

Wei, Guanghui; Li, Dong; Zuo, Qisheng; Zhang, Yani; Zhu, Rui; Zhang, Lei; Liu, Zhiyong; Qiu, Fenglong; Li, Bichun

2014-08-01

The aim of this study was to determine the activity region of Oct4 (octamer-binding transcription factor 4) promoter in Xuhuai goat, and to investigate the effect of TSA (trichostatin A) and VPA(valproicacid) on Oct4 promoter activity. Specific PCR primers of Oct4 promoter including different lengths of fragments were designed by Primer 5.0, then were amplified and cloned into PGL3-Bacic luciferase reporter vector. All the reconstruction vectors were transfected into gEF, P19 and COS7 cells, respectively. After TSA and VPA treatment, the activity of dual-luciferase reporter gene in these three transfected cells was detected. In addition, the CMV promoter of pEGFP-N1 was replaced by the -1516─+30 bp fragment of Oct4 promoter, GFP fluorescence was used to detect the activity of Oct4 promoter. The results indicated that different fragments of Oct4 promoter showed different degrees of activity in gEF, P19 and COS7 cells, and the maximal activity region of Oct4 promoter was -1516─+30 bp, the basal activity region was -238─+30 bp. Positive regulatory domains existed in the region of -1516─-946 bp and -615─-96 bp, while negative regulatory domains existed in the region of -1936─-1516 bp and -946─-615 bp. The optimum induction concentration to enhance the activity of Oct4 promoter was 1 μmol/L of TSA and 4 mmol/L of VPA. The GFP expression can be started by the fragment of -1516─+30 bp. This study provides an experimental basis for revealing the mechanism of expression and regulation of Oct4 in goat.

Overexpression of OCT4A ortholog elevates endogenous XIST in porcine parthenogenic blastocysts.

PubMed

Hwang, Jae Yeon; Choi, Kwang-Hwan; Lee, Dong-Kyung; Kim, Seung-Hun; Kim, Eun Bae; Hyun, Sang-Hwan; Lee, Chang-Kyu

2015-01-01

X-chromosome inactivation (XCI) is an epigenetic process that equalizes expression of X-borne genes between male and female eutherians. This process is observed in early eutherian embryo development in a species-specific manner. Until recently, various pluripotent factors have been suggested to regulate the process of XCI by repressing XIST expression, which is the master inducer for XCI. Recent insights into the process and its regulation have been restricted in mouse species despite the evolutionary diversity of the process and molecular mechanism among the species. OCT4A is one of the represented pluripotent factors, the gate-keeper for maintaining pluripotency, and an XIST repressor. Therefore, in here, we examined the relation between OCT4A and X-linked genes in porcine preimplantation embryos. Three X-linked genes, XIST, LOC102165544, and RLIM, were selected in present study because their orthologues have been known to regulate XCI in mice. Expression levels of OCT4A were positively correlated with XIST and LOC102165544 in female blastocysts. Furthermore, overexpression of exogenous human OCT4A in cleaved parthenotes generated blastocysts with increased XIST expression levels. However, increased XIST expression was not observed when exogenous OCT4A was obtained from early blastocysts. These results suggest the possibility that OCT4A would be directly or indirectly involved in XIST expression in earlier stage porcine embryos rather than blastocysts.

Manganese Superoxide Dismutase Gene Expression Is Induced by Nanog and Oct4, Essential Pluripotent Stem Cells' Transcription Factors.

PubMed

Solari, Claudia; Vázquez Echegaray, Camila; Cosentino, María Soledad; Petrone, María Victoria; Waisman, Ariel; Luzzani, Carlos; Francia, Marcos; Villodre, Emilly; Lenz, Guido; Miriuka, Santiago; Barañao, Lino; Guberman, Alejandra

2015-01-01

Pluripotent stem cells possess complex systems that protect them from oxidative stress and ensure genomic stability, vital for their role in development. Even though it has been reported that antioxidant activity diminishes along stem cell differentiation, little is known about the transcriptional regulation of the involved genes. The reported modulation of some of these genes led us to hypothesize that some of them could be regulated by the transcription factors critical for self-renewal and pluripotency in embryonic stem cells (ESCs) and in induced pluripotent stem cells (iPSCs). In this work, we studied the expression profile of multiple genes involved in antioxidant defense systems in both ESCs and iPSCs. We found that Manganese superoxide dismutase gene (Mn-Sod/Sod2) was repressed during diverse differentiation protocols showing an expression pattern similar to Nanog gene. Moreover, Sod2 promoter activity was induced by Oct4 and Nanog when we performed a transactivation assay using two different reporter constructions. Finally, we studied Sod2 gene regulation by modulating the expression of Oct4 and Nanog in ESCs by shRNAs and found that downregulation of any of them reduced Sod2 expression. Our results indicate that pluripotency transcription factors positively modulate Sod2 gene transcription.

Manganese Superoxide Dismutase Gene Expression Is Induced by Nanog and Oct4, Essential Pluripotent Stem Cells’ Transcription Factors

PubMed Central

Solari, Claudia; Vázquez Echegaray, Camila; Cosentino, María Soledad; Petrone, María Victoria; Waisman, Ariel; Luzzani, Carlos; Francia, Marcos; Villodre, Emilly; Lenz, Guido; Miriuka, Santiago; Barañao, Lino; Guberman, Alejandra

2015-01-01

Pluripotent stem cells possess complex systems that protect them from oxidative stress and ensure genomic stability, vital for their role in development. Even though it has been reported that antioxidant activity diminishes along stem cell differentiation, little is known about the transcriptional regulation of the involved genes. The reported modulation of some of these genes led us to hypothesize that some of them could be regulated by the transcription factors critical for self-renewal and pluripotency in embryonic stem cells (ESCs) and in induced pluripotent stem cells (iPSCs). In this work, we studied the expression profile of multiple genes involved in antioxidant defense systems in both ESCs and iPSCs. We found that Manganese superoxide dismutase gene (Mn-Sod/Sod2) was repressed during diverse differentiation protocols showing an expression pattern similar to Nanog gene. Moreover, Sod2 promoter activity was induced by Oct4 and Nanog when we performed a transactivation assay using two different reporter constructions. Finally, we studied Sod2 gene regulation by modulating the expression of Oct4 and Nanog in ESCs by shRNAs and found that downregulation of any of them reduced Sod2 expression. Our results indicate that pluripotency transcription factors positively modulate Sod2 gene transcription. PMID:26642061

Association between EML4-ALK fusion gene and thymidylate synthase mRNA expression in non-small cell lung cancer tissues

PubMed Central

XU, CHUN-WEI; WANG, GANG; WANG, WU-LONG; GAO, WEN-BIN; HAN, CHUAN-JUN; GAO, JING-SHAN; ZHANG, LI-YING; LI, YANG; WANG, LIN; ZHANG, YU-PING; TIAN, YU-WANG; QI, DONG-DONG

2015-01-01

This study aimed to investigate the association of the mRNA expression of the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene with that of thymidylate synthase (TYMS) in non-small cell lung cancer (NSCLC) tissues. Quantitative polymerase chain reaction was used to detect the expression of EML4-ALK fusion gene and TYMS mRNA in 257 cases of NSCLC. The positive rate of EML4-ALK fusion gene was 4.28% in the NSCLC tissues (11/257), and was higher in nonsmokers than in smokers (P<0.05); TYMS mRNA expression was detected in 63.42% (163/257) of cases. An association of the EML4-ALK fusion gene with TYMS expression was detected; a low expression level of TYMS mRNA was observed more frequently when the EML4-ALK fusion gene was present than when it was not detected (P<0.05). In conclusion, patients positive for the EML4-ALK fusion gene in NSCLC tissues are likely to have a low expression level of TYMS, and may benefit from the first-line chemotherapy drug pemetrexed. PMID:26136951

Association between EML4-ALK fusion gene and thymidylate synthase mRNA expression in non-small cell lung cancer tissues.

PubMed

Xu, Chun-Wei; Wang, Gang; Wang, Wu-Long; Gao, Wen-Bin; Han, Chuan-Jun; Gao, Jing-Shan; Zhang, Li-Ying; Li, Yang; Wang, Lin; Zhang, Yu-Ping; Tian, Yu-Wang; Qi, Dong-Dong

2015-06-01

This study aimed to investigate the association of the mRNA expression of the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene with that of thymidylate synthase (TYMS) in non-small cell lung cancer (NSCLC) tissues. Quantitative polymerase chain reaction was used to detect the expression of EML4-ALK fusion gene and TYMS mRNA in 257 cases of NSCLC. The positive rate of EML4-ALK fusion gene was 4.28% in the NSCLC tissues (11/257), and was higher in nonsmokers than in smokers (P<0.05); TYMS mRNA expression was detected in 63.42% (163/257) of cases. An association of the EML4-ALK fusion gene with TYMS expression was detected; a low expression level of TYMS mRNA was observed more frequently when the EML4-ALK fusion gene was present than when it was not detected (P<0.05). In conclusion, patients positive for the EML4-ALK fusion gene in NSCLC tissues are likely to have a low expression level of TYMS, and may benefit from the first-line chemotherapy drug pemetrexed.

Isolation of Oct4-Expressing Extraembryonic Endoderm Precursor Cell Lines

PubMed Central

Debeb, Bisrat G.; Galat, Vasiliy; Epple-Farmer, Jessica; Iannaccone, Steve; Woodward, Wendy A.; Bader, Michael; Iannaccone, Philip; Binas, Bert

2009-01-01

Background The extraembryonic endoderm (ExEn) defines the yolk sac, a set of membranes that provide essential support for mammalian embryos. Recent findings suggest that the committed ExEn precursor is present already in the embryonic Inner Cell Mass (ICM) as a group of cells that intermingles with the closely related epiblast precursor. All ICM cells contain Oct4, a key transcription factor that is first expressed at the morula stage. In vitro, the epiblast precursor is most closely represented by the well-characterized embryonic stem (ES) cell lines that maintain the expression of Oct4, but analogous ExEn precursor cell lines are not known and it is unclear if they would express Oct4. Methodology/Principal Findings Here we report the isolation and characterization of permanently proliferating Oct4-expressing rat cell lines (“XEN-P cell lines”), which closely resemble the ExEn precursor. We isolated the XEN-P cell lines from blastocysts and characterized them by plating and gene expression assays as well as by injection into embryos. Like ES cells, the XEN-P cells express Oct4 and SSEA1 at high levels and their growth is stimulated by leukemia inhibitory factor, but instead of the epiblast determinant Nanog, they express the ExEn determinants Gata6 and Gata4. Further, they lack markers characteristic of the more differentiated primitive/visceral and parietal ExEn stages, but exclusively differentiate into these stages in vitro and contribute to them in vivo. Conclusions/Significance Our findings (i) suggest strongly that the ExEn precursor is a self-renewable entity, (ii) indicate that active Oct4 gene expression (transcription plus translation) is part of its molecular identity, and (iii) provide an in vitro model of early ExEn differentiation. PMID:19784378

Cardiomyocyte differentiation of rat bone marrow multipotent progenitor cells is associated with downregulation of Oct-4 expression.

PubMed

Lu, Tiewei; Pelacho, Beatriz; Hao, Hong; Luo, Min; Zhu, Jing; Verfaillie, Catherine M; Tian, Jie; Liu, Zhenguo

2010-10-01

This study was to determine if bone marrow multipotent adult progenitor cells (MAPCs) underwent cardiac specification and Oct-4 expression during their cardiomyocyte differentiation in vitro. MAPCs were isolated from rat bone marrow, treated with 5-azacytidine (5-aza, 1μM) for 24h, and cultured in a serum-free medium for cardiac differentiation for up to 35 days. The cells started to express early cardiac-specific genes Nkx2.5 and GATA-4 with a significant increase in their mRNA level within 24h after 5-aza treatment. Western blotting analysis and immunofluorescence staining revealed that the cardiac-specific proteins connexin-43 and troponin I were expressed in the cells 7 days after 5-aza treatment. Flow cytometry analysis demonstrated that over 37% of the cells were positive for troponin I by 35 days of differentiation, although the cells did not display spontaneous contraction. On the other hand, the undifferentiated MAPCs expressed a significant level of the stem-cell-specific marker Oct-4 that was dramatically decreased in the cells shortly after the initiation of cardiomyocyte differentiation as evaluated using real-time (RT)-polymerase chain reaction, Western blotting, immunofluorescence staining, and flow cytometry. These data indicated that MAPCs were able to effectively differentiate into cardiomyocyte-like cells after 5-aza induction in association with downregulation of Oct-4 expression.

The HPV16 E7 oncoprotein increases the expression of Oct3/4 and stemness-related genes and augments cell self-renewal

DOE Office of Scientific and Technical Information (OSTI.GOV)

Organista-Nava, Jorge; Gómez-Gómez, Yazmín

Oct3/4 is a transcription factor involved in maintenance of the pluripotency and self-renewal of stem cells. The E7 oncoprotein and 17β-estradiol (E{sub 2}) are key factors in cervical carcinogenesis. In the present study, we aimed to investigate the effect of the HPV16 E7 oncoprotein and E{sub 2} on the expression pattern of Oct3/4, Sox2, Nanog and Fgf4. We also determined whether the E7 oncoprotein is associated with cell self-renewal. The results showed that Oct3/4, Sox2, Nanog and Fgf4 were upregulated by the E7 oncoprotein in vivo and in vitro and implicate E{sub 2} in the upregulation of these factors inmore » vivo. We also demonstrated that E7 is involved in cell self-renewal, suggesting that the HPV16 E7 oncoprotein upregulates Oct3/4, Sox2, Nanog and Fgf4 expression to maintain the self-renewal capacity of cancer stem cells. -- Graphical abstract: The HPV16 E7 oncoprotein and 17β-estradiol are involved in the upregulation of Oct3/4, Sox2, Nanog and Fgf4 expression to maintain the self-renewal ability of cancer stem cells in cervical cancer. - Highlights: •The HPV16 E7 oncoprotein enhances cellular proliferation and dedifferentiation. •The E7 oncoprotein induces stemness-related genes expression in vivo and in vitro. •The 17β-estradiol induces stemness-related genes expression in vivo. •The HPV16 E7 oncoprotein is involved in the cell self-renewal of cancer cells.« less

A defined Oct4 level governs cell state transitions of pluripotency entry and differentiation into all embryonic lineages.

PubMed

Radzisheuskaya, Aliaksandra; Chia, Gloryn Le Bin; dos Santos, Rodrigo L; Theunissen, Thorold W; Castro, L Filipe C; Nichols, Jennifer; Silva, José C R

2013-06-01

Oct4 is considered a master transcription factor for pluripotent cell self-renewal, but its biology remains poorly understood. Here, we investigated the role of Oct4 using the process of induced pluripotency. We found that a defined embryonic stem cell (ESC) level of Oct4 is required for pluripotency entry. However, once pluripotency is established, the Oct4 level can be decreased up to sevenfold without loss of self-renewal. Unexpectedly, cells constitutively expressing Oct4 at an ESC level robustly differentiated into all embryonic lineages and germline. In contrast, cells with low Oct4 levels were deficient in differentiation, exhibiting expression of naive pluripotency genes in the absence of pluripotency culture requisites. The restoration of Oct4 expression to an ESC level rescued the ability of these to restrict naive pluripotent gene expression and to differentiate. In conclusion, a defined Oct4 level controls the establishment of naive pluripotency as well as commitment to all embryonic lineages.

Gene expression in scrapie. Cloning of a new scrapie-responsive gene and the identification of increased levels of seven other mRNA transcripts.

PubMed

Dandoy-Dron, F; Guillo, F; Benboudjema, L; Deslys, J P; Lasmézas, C; Dormont, D; Tovey, M G; Dron, M

1998-03-27

To define genes associated with or responsible for the neurodegenerative changes observed in transmissible spongiform encephalopathies, we analyzed gene expression in scrapie-infected mouse brain using "mRNA differential display." The RNA transcripts of eight genes were increased 3-8-fold in the brains of scrapie-infected animals. Five of these genes have not previously been reported to exhibit increased expression in this disease: cathepsin S, the C1q B-chain of complement, apolipoprotein D, and two previously unidentified genes denominated scrapie-responsive gene (ScRG)-1 and ScRG-2, which are preferentially expressed in brain tissue. Increased expression of the three remaining genes, beta2 microglobulin, F4/80, and metallothionein II, has previously been reported to occur in experimental scrapie. Kinetic analysis revealed a concomitant increase in the levels of ScRG-1, cathepsin S, the C1q B-chain of complement, and beta2 microglobulin mRNA as well as glial fibrillary acidic protein and F4/80 transcripts, markers of astrocytosis and microglial activation, respectively. In contrast, the level of ScRG-2, apolipoprotein D, and metallothionein II mRNA was only increased at the terminal stage of the disease. ScRG-1 mRNA was found to be preferentially expressed in glial cells and to code for a short protein of 47 amino acids with a strong hydrophobic N-terminal region.

The zebrafish spiel-ohne-grenzen (spg) gene encodes the POU domain protein Pou2 related to mammalian Oct4 and is essential for formation of the midbrain and hindbrain, and for pre-gastrula morphogenesis.

PubMed

Burgess, Shawn; Reim, Gerlinde; Chen, Wenbiao; Hopkins, Nancy; Brand, Michael

2002-02-01

In early embryonic development, the brain is divided into three main regions along the anteroposterior axis: the forebrain, midbrain and hindbrain. Through retroviral insertional mutagenesis and chemical mutagenesis experiments in zebrafish, we have isolated mutations that cause abnormal hindbrain organization and a failure of the midbrain-hindbrain boundary (MHB) to form, a region that acts as an organizer for the adjacent brain regions. The mutations fail to complement the spiel-ohne-grenzen (spg) mutation, which causes a similar phenotype, but for which the affected gene is unknown. We show through genetic mapping, cloning of the proviral insertion site and allele sequencing that spg mutations disrupt pou2, a gene encoding the Pou2 transcription factor. Based on chromosomal synteny, phylogenetic sequence comparison, and expression and functional data, we suggest that pou2 is the zebrafish ortholog of mouse Oct3/Oct4 and human POU5F1. For the mammalian genes, a function in brain development has so far not been described. In the absence of functional pou2, expression of markers for the midbrain, MHB and the hindbrain primordium (pax2.1, wnt1, krox20) are severely reduced, correlating with the neuroectoderm-specific expression phase of pou2. Injection of pou2 mRNA restores these defects in spg mutant embryos, but does not activate these markers ectopically, demonstrating a permissive role for pou2. Injections of pou2-morpholinos phenocopy the spg phenotype at low concentration, further proving that spg encodes pou2. Two observations suggest that pou2 has an additional earlier function: higher pou2-morpholino concentrations specifically cause a pre-gastrula arrest of cell division and morphogenesis, and expression of pou2 mRNA itself is reduced in spg-homozygous embryos at this stage. These experiments suggest two roles for pou2. Initially, Pou2 functions during early proliferation and morphogenesis of the blastomeres, similar to Oct3/4 in mammals during formation

OCT4 expression mediates partial cardiomyocyte reprogramming of mesenchymal stromal cells

PubMed Central

Montanari, Sonia; Santa-Cruz, Diego; Viswanathan, Sowmya; Keating, Armand

2017-01-01

Mesenchymal stem/stromal cells (MSCs) are in numerous cell therapy clinical trials, including for injured myocardium. Acquisition of cardiomyocyte characteristics by MSCs may improve cardiac regeneration but the mechanisms regulating this process are unclear. Here, we investigated whether the pluripotency transcription factor OCT4 is involved in the activation of cardiac lineage genetic programs in MSCs. We employed our established co-culture model of MSCs with rat embryonic cardiomyocytes showing co-expression of cardiac markers on MSCs independent of cell fusion. Bone marrow-derived MSCs were isolated from transgenic mice expressing GFP under the control of the cardiac-specific α-myosin heavy chain promoter. After 5 days of co-culture, MSCs expressed cardiac specific genes, including Nkx2.5, atrial natriuretic factor and α-cardiac actin. The frequency of GFP+ cells was 7.6±1.9%, however, these cells retained the stromal cell phenotype, indicating, as expected, only partial differentiation. Global OCT4 expression increased 2.6±0.7-fold in co-cultured MSCs and of interest, 87±5% vs 79±4% of MSCs expressed OCT4 by flow cytometry in controls and after co-culture, respectively. Consistent with the latter observation, the GFP+ cells did not express nuclear OCT4 and showed a significant increase in OCT4 promoter methylation compared with undifferentiated MSCs (92% vs 45%), inferring that OCT4 is regulated by an epigenetic mechanism. We further showed that siRNA silencing of OCT4 in MSCs resulted in a reduced frequency of GFP+ cells in co-culture to less than 1%. Our data infer that OCT4 expression may have a direct effect on partial cardiomyocyte reprogramming of MSCs and suggest a new mechanism(s) associated with MSC multipotency and a requirement for crosstalk with the cardiac microenvironment. PMID:29216265

OCT4 expression mediates partial cardiomyocyte reprogramming of mesenchymal stromal cells.

PubMed

Yannarelli, Gustavo; Pacienza, Natalia; Montanari, Sonia; Santa-Cruz, Diego; Viswanathan, Sowmya; Keating, Armand

2017-01-01

Mesenchymal stem/stromal cells (MSCs) are in numerous cell therapy clinical trials, including for injured myocardium. Acquisition of cardiomyocyte characteristics by MSCs may improve cardiac regeneration but the mechanisms regulating this process are unclear. Here, we investigated whether the pluripotency transcription factor OCT4 is involved in the activation of cardiac lineage genetic programs in MSCs. We employed our established co-culture model of MSCs with rat embryonic cardiomyocytes showing co-expression of cardiac markers on MSCs independent of cell fusion. Bone marrow-derived MSCs were isolated from transgenic mice expressing GFP under the control of the cardiac-specific α-myosin heavy chain promoter. After 5 days of co-culture, MSCs expressed cardiac specific genes, including Nkx2.5, atrial natriuretic factor and α-cardiac actin. The frequency of GFP+ cells was 7.6±1.9%, however, these cells retained the stromal cell phenotype, indicating, as expected, only partial differentiation. Global OCT4 expression increased 2.6±0.7-fold in co-cultured MSCs and of interest, 87±5% vs 79±4% of MSCs expressed OCT4 by flow cytometry in controls and after co-culture, respectively. Consistent with the latter observation, the GFP+ cells did not express nuclear OCT4 and showed a significant increase in OCT4 promoter methylation compared with undifferentiated MSCs (92% vs 45%), inferring that OCT4 is regulated by an epigenetic mechanism. We further showed that siRNA silencing of OCT4 in MSCs resulted in a reduced frequency of GFP+ cells in co-culture to less than 1%. Our data infer that OCT4 expression may have a direct effect on partial cardiomyocyte reprogramming of MSCs and suggest a new mechanism(s) associated with MSC multipotency and a requirement for crosstalk with the cardiac microenvironment.

The B-Cell Specific Transcription Factor, Oct-2, Promotes Epstein-Barr Virus Latency by Inhibiting the Viral Immediate-Early Protein, BZLF1

PubMed Central

Robinson, Amanda R.; Kwek, Swee Sen; Kenney, Shannon C.

2012-01-01

The Epstein-Barr virus (EBV) latent-lytic switch is mediated by the BZLF1 immediate-early protein. EBV is normally latent in memory B cells, but cellular factors which promote viral latency specifically in B cells have not been identified. In this report, we demonstrate that the B-cell specific transcription factor, Oct-2, inhibits the function of the viral immediate-early protein, BZLF1, and prevents lytic viral reactivation. Co-transfected Oct-2 reduces the ability of BZLF1 to activate lytic gene expression in two different latently infected nasopharyngeal carcinoma cell lines. Furthermore, Oct-2 inhibits BZLF1 activation of lytic EBV promoters in reporter gene assays, and attenuates BZLF1 binding to lytic viral promoters in vivo. Oct-2 interacts directly with BZLF1, and this interaction requires the DNA-binding/dimerization domain of BZLF1 and the POU domain of Oct-2. An Oct-2 mutant (Δ262–302) deficient for interaction with BZLF1 is unable to inhibit BZLF1-mediated lytic reactivation. However, an Oct-2 mutant defective for DNA-binding (Q221A) retains the ability to inhibit BZLF1 transcriptional effects and DNA-binding. Importantly, shRNA-mediated knockdown of endogenous Oct-2 expression in several EBV-positive Burkitt lymphoma and lymphoblastoid cell lines increases the level of lytic EBV gene expression, while decreasing EBNA1 expression. Moreover, treatments which induce EBV lytic reactivation, such as anti-IgG cross-linking and chemical inducers, also decrease the level of Oct-2 protein expression at the transcriptional level. We conclude that Oct-2 potentiates establishment of EBV latency in B cells. PMID:22346751

Squamous Cell Carcinoma Antigen-encoding Genes SERPINB3/B4 as Potentially Useful Markers for the Stratification of HNSCC Tumours.

PubMed

Saidak, Zuzana; Morisse, Mony Chenda; Chatelain, Denis; Sauzay, Chloé; Houessinon, Aline; Guilain, Nelly; Soyez, Marion; Chauffert, Bruno; Dakpé, Stéphanie; Galmiche, Antoine

2018-03-01

The squamous cell carcinoma antigen (SCCA), encoded by the genes SERPINB3/B4, is a tumour marker produced by head and neck squamous cell carcinoma (HNSCC). We aimed to examine SERPINB3/B4 mRNA levels and its clinical significance in the therapeutic context. We retrieved mRNA expression levels, clinical, pathological and genomic data for 520 HNSCC from The Cancer Genome Atlas (TCGA). HNSCC tumours express high levels of SERPINB3/B4 mRNA. SERPINB3 expression differs depending on Human papillomavirus (HPV) infection status, primary tumour location, grade and differentiation, extension to lymph nodes and extracapsular spread. Interestingly, we observed an association between SERPINB3/B4 and the presence of tumour immune infiltrate as well as the expression of the immune checkpoint regulators PD-L1/PD-L2 that depended on HPV status. Our findings point to potential interest of SERPINB3/B4 for the stratification of HNSCC patients in the therapeutic context. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

[Correlations between OCT4 expression and clinicopathological factors and prognosis of patients with lung adenocarcinoma].

PubMed

Zhang, Xueyan; Wang, Huimin; Jin, Bo; Dong, Qianggang; Huang, Jinsu; Han, Baohui

2013-04-01

In recent years, cases of lung adenocarcinoma morbidity have consistently grown. OCT4 is the key gene that controls the automatic renewal of stem cells, and regulates the proliferation and differentiation of cancer stem cells. The aim of this study is to detect OCT4 expression in lung adenocarcinoma tissues, and to evaluate its relevance in the metastasis, chemotherapeutic effect, and prognosis in lung adenocarcinoma patients. Immunofluorescence method was employed to detect OCT4 expression in lung adenocarcinoma tissues. The relationship between OCT4 expression and clinical pathological indicators is examined through chi-square test. Moreover, the survival rate is calculated through the Kaplan-Meier survivorship curve. Finally, the relevance between the indicators and patient survival is estimated using Cox analysis. Among the 126 tissue samples of lung adenocarcinoma, 91 showed OCT4 positive cells. OCT4 expression is closely related to metastasis and chemoresistance in lung adenocarcinoma patients, and negatively corresponds to the patients' disease-free survival and survival periods. OCT4 expression is related to metastasis and chemoresistance in lung adenocarcinoma patients, and thus indicates poor prognosis.

Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation

PubMed Central

Mora-Castilla, Sergio; Tejedo, Juan R.; Díaz, Irene; Hitos, Ana B.; Cahuana, Gladys M.; Hmadcha, Abdelkrim; Martín, Franz; Soria, Bernat

2014-01-01

The function of pluripotency genes in differentiation is a matter of investigation. We report here that Nanog and Oct4 are reexpressed in two mouse embryonic stem cell (mESC) lines following exposure to the differentiating agent DETA/NO. Both cell lines express a battery of both endoderm and mesoderm markers following induction of differentiation with DETA/NO-based protocols. Confocal analysis of cells undergoing directed differentiation shows that the majority of cells expressing Nanog express also endoderm genes such as Gata4 and FoxA2 (75.4% and 96.2%, resp.). Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment. Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression. Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transition. PMID:25544848

The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

PubMed Central

Fong, Yick W; Ho, Jaclyn J; Inouye, Carla; Tjian, Robert

2014-01-01

Acquisition of pluripotency is driven largely at the transcriptional level by activators OCT4, SOX2, and NANOG that must in turn cooperate with diverse coactivators to execute stem cell-specific gene expression programs. Using a biochemically defined in vitro transcription system that mediates OCT4/SOX2 and coactivator-dependent transcription of the Nanog gene, we report the purification and identification of the dyskerin (DKC1) ribonucleoprotein complex as an OCT4/SOX2 coactivator whose activity appears to be modulated by a subset of associated small nucleolar RNAs (snoRNAs). The DKC1 complex occupies enhancers and regulates the expression of key pluripotency genes critical for self-renewal in embryonic stem (ES) cells. Depletion of DKC1 in fibroblasts significantly decreased the efficiency of induced pluripotent stem (iPS) cell generation. This study thus reveals an unanticipated transcriptional role of the DKC1 complex in stem cell maintenance and somatic cell reprogramming. DOI: http://dx.doi.org/10.7554/eLife.03573.001 PMID:25407680

Pontin functions as an essential coactivator for Oct4-dependent lincRNA expression in mouse embryonic stem cells.

PubMed

Boo, Kyungjin; Bhin, Jinhyuk; Jeon, Yoon; Kim, Joomyung; Shin, Hi-Jai R; Park, Jong-Eun; Kim, Kyeongkyu; Kim, Chang Rok; Jang, Hyonchol; Kim, In-Hoo; Kim, V Narry; Hwang, Daehee; Lee, Ho; Baek, Sung Hee

2015-04-10

The actions of transcription factors, chromatin modifiers and noncoding RNAs are crucial for the programming of cell states. Although the importance of various epigenetic machineries for controlling pluripotency of embryonic stem (ES) cells has been previously studied, how chromatin modifiers cooperate with specific transcription factors still remains largely elusive. Here, we find that Pontin chromatin remodelling factor plays an essential role as a coactivator for Oct4 for maintenance of pluripotency in mouse ES cells. Genome-wide analyses reveal that Pontin and Oct4 share a substantial set of target genes involved in ES cell maintenance. Intriguingly, we find that the Oct4-dependent coactivator function of Pontin extends to the transcription of large intergenic noncoding RNAs (lincRNAs) and in particular linc1253, a lineage programme repressing lincRNA, is a Pontin-dependent Oct4 target lincRNA. Together, our findings demonstrate that the Oct4-Pontin module plays critical roles in the regulation of genes involved in ES cell fate determination.

Role of defective Oct-2 and OCA-B expression in immunoglobulin production and Kaposi's sarcoma-associated herpesvirus lytic reactivation in primary effusion lymphoma.

PubMed

Di Bartolo, Daniel L; Hyjek, Elizabeth; Keller, Shannon; Guasparri, Ilaria; Deng, Hongyu; Sun, Ren; Chadburn, Amy; Knowles, Daniel M; Cesarman, Ethel

2009-05-01

Primary effusion lymphoma (PEL) is a distinct type of B-cell non-Hodgkin lymphoma characterized by the presence of Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8). Despite having a genotype and gene expression signature of highly differentiated B cells, PEL does not usually express surface or cytoplasmic immunoglobulin (Ig). We show the lack of Oct-2 and OCA-B transcription factors to be responsible, at least in part, for this defect in Ig production. Like Ig genes, ORF50, the key regulator of the switch from latency to lytic reactivation, contains an octamer motif within its promoter. We therefore examined the impact of Oct-2 and OCA-B on ORF50 activation. The binding of Oct-1 to the ORF50 promoter has been shown to significantly enhance ORF50 transactivation. We found that Oct-2, on the other hand, inhibited ORF50 expression and consequently lytic reactivation by competing with Oct-1 for the octamer motif in the ORF50 promoter. Our data suggest that Oct-2 downregulation in infected cells would be favorable to KSHV in allowing for efficient viral reactivation.

Nitric oxide enhances Oct-4 expression in bone marrow stem cells and promotes endothelial differentiation.

PubMed

Chu, Ling; Jiang, Yuehua; Hao, Hong; Xia, Yong; Xu, Jian; Liu, Zehao; Verfaillie, Catherine M; Zweier, Jay L; Liu, Zhenguo

2008-09-04

This study was designed to investigate the role of nitric oxide (NO) in bone marrow stem cells and their differentiation into endothelial cells in vitro. Adult mouse bone marrow multipotent progenitor cells (MAPCs) were used as the source of stem cells. Oct-4 expression (both mRNA and protein) was significantly increased by up to 68.0% in MAPCs when incubated with NO donors DETA-NONOate or sodium nitroprusside (SNP) in a concentration-dependant manner (n=3, P<0.05). However, the cell proliferation was dramatically decreased by over 3-folds when treated with DETA-NONOate or SNP for 48 h (n=3, P<0.05). When MAPCs were exposed to DETA-NONOate (100 microM) for the first 48 h during differentiation, the expression (both mRNA and protein) of vWF was significantly increased at day 14 in the differentiating cells. The effects of DETA-NONOate or SNP on cell proliferation, Oct-4 expression and endothelial differentiation of MAPCs were not affected by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one or cGMP analog 8-Br-cGMP. These data indicate that NO may regulate both the pluripotency and differentiation of MAPCs via a cGMP-independent mechanism.

Differential expression of Oct4 in HPV-positive and HPV-negative cervical cancer cells is not regulated by DNA methyltransferase 3A.

PubMed

Liu, Dongbo; Zhou, Peng; Zhang, Li; Wu, Gengze; Zheng, Yingru; He, Fengtian

2011-10-01

The colony-forming ability of cervical cancer is affected by many factors. Oct4, an important transcription factor, is highly expressed in several tumors and promotes the colony-forming ability of cancer cells. Thus, it is considered a potential target for the treatment of cancer. However, we know little about the expression level of Oct4 and its epigenetic regulatory mechanism in cervical cancer cells. In this study, we are the first to observe that human papillomavirus (HPV)-positive cervical cancer cell lines (HeLa, Caski) have a stronger colony-forming ability than HPV-negative cervical cancer cell lines (C-33A). Moreover, the expression level of Oct4 in both HeLa and Caski cells was also higher than that in C-33A cells. We then confirmed that there was a negative correlation between the expression of Oct4 and DNMT3A in these three types of cervical cancer cells, whereas DNA methyltransferase 1 and 3B had no differences among the cell lines. However, after DNA methylation in both key regulatory regions of the Oct4 gene and the genomic levels were analyzed, we found that DNA methyltransferase 3A could neither regulate the expression of Oct4 nor affect the whole level of genomic DNA methylation. These results suggest three points: (1) Oct4 might be treated as a new target for the treatment of cervical cancer, (2) we could not inhibit the expression of Oct4 by DNA demethylation, and (3) HPV virus might initiate cervical carcinogenesis by upregulation of Oct4 expression.

Structural Mechanism behind Distinct Efficiency of Oct4/Sox2 Proteins in Differentially Spaced DNA Complexes

PubMed Central

Yesudhas, Dhanusha; Anwar, Muhammad Ayaz; Panneerselvam, Suresh; Durai, Prasannavenkatesh; Shah, Masaud; Choi, Sangdun

2016-01-01

The octamer-binding transcription factor 4 (Oct4) and sex-determining region Y (SRY)-box 2 (Sox2) proteins induce various transcriptional regulators to maintain cellular pluripotency. Most Oct4/Sox2 complexes have either 0 base pairs (Oct4/Sox20bp) or 3 base pairs (Oct4/Sox23bp) separation between their DNA-binding sites. Results from previous biochemical studies have shown that the complexes separated by 0 base pairs are associated with a higher pluripotency rate than those separated by 3 base pairs. Here, we performed molecular dynamics (MD) simulations and calculations to determine the binding free energy and per-residue free energy for the Oct4/Sox20bp and Oct4/Sox23bp complexes to identify structural differences that contribute to differences in induction rate. Our MD simulation results showed substantial differences in Oct4/Sox2 domain movements, as well as secondary-structure changes in the Oct4 linker region, suggesting a potential reason underlying the distinct efficiencies of these complexes during reprogramming. Moreover, we identified key residues and hydrogen bonds that potentially facilitate protein-protein and protein-DNA interactions, in agreement with previous experimental findings. Consequently, our results confess that differential spacing of the Oct4/Sox2 DNA binding sites can determine the magnitude of transcription of the targeted genes during reprogramming. PMID:26790000

Nitrative DNA damage and Oct3/4 expression in urinary bladder cancer with Schistosomahaematobium infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Ning; Thanan, Raynoo; Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie

Highlights: {yields} Oct3/4-positive cells increase in Schistosoma haematobium (SH)-associated bladder cancer. {yields} iNOS-dependent DNA lesion, 8-nitroguanine, was formed in Oct3/4-positive cells. {yields} 8-Nitroguanine formed in stem-like cells plays a role in SH-induced carcinogenesis. {yields} Mutant stem cells may participate in inflammation-related carcinogenesis. -- Abstract: To investigate whether mutant stem cells participate in inflammation-related carcinogenesis, we performed immunohistochemical analysis to examine nitrative and oxidative DNA lesions (8-nitroguanine and 8-oxodG) and a stem cell marker Oct3/4 in bladder tissues obtained from cystitis and bladder cancer patients infected with Schistosomahaematobium (S. haematobium). We also detected the expression of nuclear factor-{kappa}B (NF-{kappa}B) and induciblemore » nitric oxide synthase (iNOS), which lead to 8-nitroguanine formation. The staining intensity of 8-nitroguanine and 8-oxodG was significantly higher in bladder cancer and cystitis tissues than in normal tissues. iNOS expression was colocalized with NF-{kappa}B in 8-nitroguanine-positive tumor cells from bladder cancer patients. Oct3/4 expression was significantly increased in cells from S. haematobium-associated bladder cancer tissues in comparison to normal bladder and cancer tissues without infection. Oct3/4 was also expressed in epithelial cells of cystitis patients. Moreover, 8-nitroguanine was formed in Oct3/4-positive stem cells in S. haematobium-associated cystitis and cancer tissues. In conclusion, inflammation by S.haematobium infection may increase the number of mutant stem cells, in which iNOS-dependent DNA damage occurs via NF-{kappa}B activation, leading to tumor development.« less

Influence of culture medium composition on relative mRNA abundances in domestic cat embryos.

PubMed

Hribal, R; Jewgenow, K; Braun, B C; Comizzoli, P

2013-04-01

Different culture conditions have been used to produce domestic cat embryos. As part of the in vitro procedures, the medium composition significantly affects the quality of the embryo development also. Quality assessments based on cleavage kinetics and blastomere symmetry are useful, but embryos also can differ in their relative gene expression patterns despite similar morphological characteristics. The aim of this study was to compare cat embryos produced with two different in vitro culture systems routinely used in two different laboratories [Smithsonian Conservation Biology Institute, Washington D.C., USA (SCBI) and Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany (IZW)]. Specifically, relative mRNA expression patterns of critical genes for pre-implantation embryo development were assessed in both conditions. Embryos were produced in parallel in both culture systems by IVF using frozen-thawed ejaculated semen in the United States and fresh epididymal sperm in Germany. Success of embryo development in vitro was recorded as well as relative mRNA abundances [DNA methyltransferases 1 and 3A (DNMT1, DNMT3A), gap junction protein alpha 1 (GJA1), octamer-binding transcription factor 4 [OCT4], insulin-like growth factors 1 and 2 receptors (IGF1R, IGF2R), beta-actin (ACTB)] in pools of days 4-5 morulae by semi-quantitative RT-PCR assay. Percentages of cleaved embryos were similar (p > 0.05) between both culture systems, regardless of the location. OCT4 mRNA abundance was higher (p < 0.05) in embryos derived in the SCBI culture system compared with those from the IZW system when epididymal sperm was used for IVF. No clear correlation between the expression pattern and the culture system could be found for all other genes. It is suggested that OCT4 expression might be affected by the media composition in some conditions and can be the indicator of a better embryo quality. © 2012 Blackwell Verlag GmbH.

4-D OCT in Developmental Cardiology

NASA Astrophysics Data System (ADS)

Jenkins, Michael W.; Rollins, Andrew M.

Although strong evidence exists to suggest that altered cardiac function can lead to CHDs, few studies have investigated the influential role of cardiac function and biophysical forces on the development of the cardiovascular system due to a lack of proper in vivo imaging tools. 4-D imaging is needed to decipher the complex spatial and temporal patterns of biomechanical forces acting upon the heart. Numerous solutions over the past several years have demonstrated 4-D OCT imaging of the developing cardiovascular system. This chapter will focus on these solutions and explain their context in the evolution of 4-D OCT imaging. The first sections describe the relevant techniques (prospective gating, direct 4-D imaging, retrospective gating), while later sections focus on 4-D Doppler imaging and measurements of force implementing 4-D OCT Doppler. Finally, the techniques are summarized, and some possible future directions are discussed.

Participation of OCT3/4 and beta-catenin during dysgenetic gonadal malignant transformation.

PubMed

Palma, Icela; Peña, Rocio-Yolanda; Contreras, Alejandra; Ceballos-Reyes, Guillermo; Coyote, Ninel; Eraña, Luis; Kofman-Alfaro, Susana; Queipo, Gloria

2008-05-18

Gonadoblastoma (GB) is an in situ tumor consisting of a heterogeneous population of mature and immature germ cells, other cells resembling immature Sertoli/granulosa cells, and Leydig/lutein-like cells, may also be present. GB almost exclusively affects a subset of patients with intersex disorders and in 30% of them overgrowth of the germinal component of the tumor is observed and the lesion is term dysgerminoma/seminoma. Several pathways have been proposed to explain the malignant process, and abnormal OCT3/4 expression is the most robust risk factor for malignant transformation. Some authors have suggested that OCT3/4 and beta-catenin might both be involved in the same oncogenic pathway, as both genes are master regulators of cell differentiation and, overexpression of either gene may result in cancer development. The mechanism by which beta-catenin participates in GB transformation is not completely clear and exploration of the E-cadherin pathway did not conclusively show that this pathway participated in the molecular pathogenesis of GB. Here we analyze seven patients with mixed gonadal dysgenesis and GB, in an effort to elucidate the participation of beta-catenin and E-cadherin, as well as OCT3/4, in the oncogenic pathways involved in the transformation of GB into seminoma/dysgerminoma. We conclude that the proliferation of immature germ cells in GB may be due to an interaction between OCT3/4 and accumulated beta-catenin in the nuclei of the immature germ cells.

Design and evaluation of an intraocular B-scan OCT-guided 36-gauge needle

NASA Astrophysics Data System (ADS)

Shen, Jin H.; Joos, Karen M.

2015-03-01

Optical coherence tomography imaging is widely used in ophthalmology and optometry clinics for diagnosing retinal disorders. External microscope-mounted OCT operating room systems have imaged retinal changes immediately following surgical manipulations. However, the goal is to image critical surgical maneuvers in real time. External microscope-mounted OCT systems have some limitations with problems tracking constantly moving intraocular surgical instruments, and formation of absolute shadows by the metallic surgical instruments upon the underlying tissues of interest. An intraocular OCT-imaging probe was developed to resolve these problems. A disposable 25-gauge probe tip extended beyond the handpiece, with a 36-gauge needle welded to a disposable tip with its end extending an additional 3.5 mm. A sealed 0.35 mm diameter GRIN lens protected the fiber scanner and focused the scanning beam at a 3 to 4 mm distance. The OCT engine was a very high-resolution spectral-domain optical coherence tomography (SDOCT) system (870 nm, Bioptigen, Inc. Durham, NC) which produced 2000 A-scan lines per B-scan image at a frequency of 5 Hz with the fiber optic oscillations matched to this frequency. Real-time imaging of the needle tip as it touched infrared paper was performed. The B-scan OCT-needle was capable of real-time performance and imaging of the phantom material. In the future, the B-scan OCT-guided needle will be used to perform sub-retinal injections.

Transforming growth factor-beta 1 (TGF-beta1) promotes IL-2 mRNA expression through the up-regulation of NF-kappaB, AP-1 and NF-AT in EL4 cells.

PubMed

Han, S H; Yea, S S; Jeon, Y J; Yang, K H; Kaminski, N E

1998-12-01

Transforming growth factor beta1 (TGF-beta1) has been previously shown to modulate interleukin 2 (IL-2) secretion by activated T-cells. In the present studies, we determined that TGF-beta1 induced IL-2 mRNA expression in the murine T-cell line EL4, in the absence of other stimuli. IL-2 mRNA expression was significantly induced by TGF-beta1 (0.1-1 ng/ml) over a relatively narrow concentration range, which led to the induction of IL-2 secretion. Under identical condition, we examined the effect of TGF-beta1 on the activity of nuclear factor AT (NF-AT), nuclear factor kappaB (NF-kappaB), activator protein-1 (AP-1) and octamer, all of which contribute to the regulation of IL-2 gene expression. Electrophoretic mobility shift assays showed that TGF-beta1 markedly increased NF-AT, NF-kappaB and AP-1 binding to their respective cognate DNA binding sites, whereas octamer binding remained constant, as compared with untreated cells. Employing a reporter gene expression system with p(NF-kappaB)3-CAT, p(NF-AT)3-CAT and p(AP-1)3-CAT, TGF-beta1 treatment of transfected EL4 cells induced a dose-related increase in chloramphenicol acetyltransferase activity that correlated well with the DNA binding profile found in the electrophoretic mobility shift assay studies. These results show that TGF-beta1, in the absence of any additional stimuli, up-regulates the activity of key transcription factors involved in IL-2 gene expression, including NF-AT, NF-kappaB and AP-1, to help promote IL-2 mRNA expression by EL4 cells.

Akt-mediated phosphorylation of Oct4 is associated with the proliferation of stem-like cancer cells

PubMed Central

ZHAO, QING-WEI; ZHOU, YAN-WEN; LI, WEN-XIN; KANG, BO; ZHANG, XIAO-QIAN; YANG, YING; CHENG, JIE; YIN, SHENG-YONG; TONG, YING; HE, JIAN-QIN; YAO, HANG-PING; ZHENG, MIN; WANG, YING-JIE

2015-01-01

Oct4 protein encoded by POU5F1 plays a pivotal role in maintaining the self-renewal of pluripotent stem cells; however, its presence in cancer cells remains controversial. In the present study, we provided evidence that the transcripts of authentic OCT4 gene (OCT4A) and its multiple pseudogenes were detected in a variety of cancer cell lines. A few major bands were also detected by western blotting using an anti-Oct4A monoclonal antibody. Moreover, an anti-Oct4-pT235 antibody was used to identify a band in the majority of the tested cancer cell lines that coincided with one of the anti-Oct4A bands which was decreasable by a specific shRNA. The Oct4-pT235 signals were also detected in human glioblastoma and liver cancer specimens by immunofluorescence microscopy and immunohistochemistry. U87 glioblastoma cells were cultured in a neural stem cell medium to induce the formation of neurospheres rich in stem-like cancer cells. The levels of Oct4-pT235 in the sphere cells were markedly increased compared to their monolayer parental cells, a result that was accompanied by upregulation of the PI3K-Akt pathway. Akti-1/2, a specific inhibitor of Akt, effectively reduced the level of Oct4-pT235 and attenuated the proliferation of U87 sphere cells. ITE, an agonist of the aryl hydrocarbon receptor, also significantly attenuated the Akt-mediated phosphorylation of Oct4 in glioblastoma and liver cancer cells, and reduced their tumorigenic potential in a xenograft tumor model. Taken together, we concluded that the Akt-mediated phosphorylation of Oct4A or its homolog protein was associated with the proliferation of stem-like cancer cells that may serve as a novel biomarker and drug target for certain types of cancer. PMID:25625591

Antisense targeting of 3' end elements involved in DUX4 mRNA processing is an efficient therapeutic strategy for facioscapulohumeral dystrophy: a new gene-silencing approach.

PubMed

Marsollier, Anne-Charlotte; Ciszewski, Lukasz; Mariot, Virginie; Popplewell, Linda; Voit, Thomas; Dickson, George; Dumonceaux, Julie

2016-04-15

Defects in mRNA 3'end formation have been described to alter transcription termination, transport of the mRNA from the nucleus to the cytoplasm, stability of the mRNA and translation efficiency. Therefore, inhibition of polyadenylation may lead to gene silencing. Here, we choose facioscapulohumeral dystrophy (FSHD) as a model to determine whether or not targeting key 3' end elements involved in mRNA processing using antisense oligonucleotide drugs can be used as a strategy for gene silencing within a potentially therapeutic context. FSHD is a gain-of-function disease characterized by the aberrant expression of the Double homeobox 4 (DUX4) transcription factor leading to altered pathogenic deregulation of multiple genes in muscles. Here, we demonstrate that targeting either the mRNA polyadenylation signal and/or cleavage site is an efficient strategy to down-regulate DUX4 expression and to decrease the abnormally high-pathological expression of genes downstream of DUX4. We conclude that targeting key functional 3' end elements involved in pre-mRNA to mRNA maturation with antisense drugs can lead to efficient gene silencing and is thus a potentially effective therapeutic strategy for at least FSHD. Moreover, polyadenylation is a crucial step in the maturation of almost all eukaryotic mRNAs, and thus all mRNAs are virtually eligible for this antisense-mediated knockdown strategy. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Effects of Coriolus versicolor polysaccharide B on monocyte chemoattractant protein 1 gene expression in rat.

PubMed

Song, Lie-Chang; Chen, Hai-Sheng; Lou, Ning; Song, Chang; Zeng, Jun; Fu, Ting-Huan

2002-06-01

To investigate the effect of Coriolus versicolor polysaccharide B (CVPS-B), a new water-soluble component of polysaccharides from the fungus Coriolus versicolor (Fr) L on monocyte chemoattractant protein-1 (MCP-1) gene expression in rat splenocytes. Expression of MCP-1 mRNA in rat splenocytes was examined by reverse transcription-polymerase chain reaction (RT-PCR) with beta- actin as an internal standard. Sequencing of RT-PCR products was performed to confirm their specificity in MCP-1 gene composition. (1) Without pre-treatment of lipopolysaccharide (LPS), the relative MCP-1 mRNA expression ratios (MCP-1/beta-actin) for the saline control group and for CVPS-B groups in 3 different doses (10, 20, and 30 mg . kg-1 . d-1, ip, for 4 d) were 1.4 +/- 0.3, 1.6 +/- 0.4, 1.7 +/- 0.5, and 1.5 +/- 0.4, respectively (P > 0.05); (2) LPS (10 microg . kg-1, ip) enhanced the expression of MPC-1 mRNA by the ratio of 114 %; (3) pre-treatment with CVPS-B of 4 different doses (5, 10, 30, and 50 mg . kg-1 . d-1, ip, for 4 d) decreased the LPS induced expression of MPC-1 mRNA by the ratios of 51 %, 70 %, 84 %, and 99 %, respectively (n = 6). In a dose-related fashion, CVPS-B inhibited the expression of MCP-1 mRNA induced by LPS in the rat splenocytes, but did not significantly affect the expression of MPC-1 mRNA in the normal rat.

Variations in endothelin receptor B subtype 2 (EDNRB2) coding sequences and mRNA expression levels in 4 Muscovy duck plumage colour phenotypes.

PubMed

Wu, N; Qin, H; Wang, M; Bian, Y; Dong, B; Sun, G; Zhao, W; Chang, G; Xu, Q; Chen, G

2017-04-01

1. Endothelin receptor B subtype 2 (EDNRB2) is a paralog of EDNRB, which encodes a 7-transmembrane G-protein coupled receptor. Previous studies reported that EDNRB was essential for melanoblast migration in mammals and ducks. 2. Muscovy ducks have different plumage colour phenotypes. Variations in EDNRB2 coding sequences (CDSs) and mRNA expression levels were investigated in 4 different Muscovy duck plumage colour phenotypes, including black, black mutant, silver and white head. 3. The EDNRB2 gene from Muscovy duck was cloned; it had a length of 6435 bp and encoded 437 amino acids. The coding region was screened and potential single nucleotide polymorphisms were identified. Eight mutations were obtained, including one missense variant (c.64C > T) and 7 synonymous substitutions. The substitutions were associated with plumage colour phenotypes. 4. The EDNRB2 mRNA expression levels were compared between feather pulp from black birds and black mutant birds. The results indicated that EDNRB2 transcripts in feather pulp were significantly higher in black feathers than in white feathers. 5. The results determined the variation of EDNRB2 CDS and mRNA expression in Muscovy ducks of various plumage colours.

WNT2B2 mRNA, up-regulated in primary gastric cancer, is a positive regulator of the WNT- beta-catenin-TCF signaling pathway.

PubMed

Katoh, M; Kirikoshi, H; Terasaki, H; Shiokawa, K

2001-12-21

Genetic alterations of WNT signaling molecules lead to carcinogenesis through activation of the beta-catenin-TCF signaling pathway. We have previously cloned and characterized WNT2B/WNT13 gene on human chromosome 1p13, which is homologous to proto-oncogene WNT2 on human chromosome 7q31. WNT2B1 and WNT2B2 mRNAs, generated from the WNT2B gene due to alternative splicing of the alternative promoter type, encode almost identical polypeptides with divergence in the N-terminal region. WNT2B2 mRNA rather than WNT2B1 mRNA is preferentially expressed in NT2 cells with the potential of neuronal differentiation. Here, we describe our investigations of expression of WNT2B mRNAs in various types of human primary cancer. Matched tumor/normal expression array analysis revealed that WNT2B mRNAs were significantly up-regulated in 2 of 8 cases of primary gastric cancer. WNT2B2 mRNA rather than WNT2B1 mRNA was found to be preferentially up-regulated in a case of primary gastric cancer (signet ring cell carcinoma). Function of WNT2B1 mRNA and that of WNT2B2 mRNA were investigated by using Xenopus axis duplication assay. Injection of synthetic WNT2B1 mRNA into the ventral marginal zone of fertilized Xenopus eggs at the 4-cell stage did not induce axis duplication. In contrast, ventral injection of synthetic WNT2B2 mRNA induced axis duplication in 90% of embryos (complete axis duplication, 24%). These results strongly suggest that WNT2B2 up-regulation in some cases of gastric cancer might lead to carcinogenesis through activation of the beta-catenin-TCF signaling pathway.

Selenium Deficiency Affects the mRNA Expression of Inflammatory Factors and Selenoprotein Genes in the Kidneys of Broiler Chicks.

PubMed

Zhang, Jiu-Li; Xu, Bo; Huang, Xiao-Dan; Gao, Yu-Hong; Chen, Yu; Shan, An-Shan

2016-05-01

The aim of this study was to investigate the influence of Se deficiency on the transcription of inflammatory factors and selenoprotein genes in the kidneys of broiler chicks. One hundred fifty 1-day-old broiler chicks were randomly assigned to two groups fed with either a low-Se diet (L group, 0.033 mg/kg Se) or an adequate Se diet (C group, 0.2 mg/kg Se). The levels of uric acid (UA) and creatinine (Cr) in the serum and the mRNA levels of 6 inflammatory factors and 25 selenoprotein genes in the kidneys were measured as the clinical signs of Se deficiency occurred at 20 days old. The results indicated that the contents of UA and Cr in the serum increased in L group (p < 0.05), and the mRNA levels of the inflammatory factors (NF-κB, iNOS, COX-2, and TNF-α) increased in L group (p < 0.05). Meanwhile, the mRNA levels of PTGEs and HO-1 were not changed. In addition, 25 selenoprotein transcripts displayed ubiquitous expression in the kidneys of the chicks. The mRNA levels of 14 selenoprotein genes (Dio1, Dio2, GPx3, Sepp1, SelH, SelI, SelK, Sepn1, SelO, SelW, Sep15, SelT, SelU, and SelS) decreased, and 9 selenoprotein genes (GPx1, GPx2, GPx4, SelPb, Txnrd1, Txnrd2, Txnrd3, SPS2, and SelM) increased in L group (p < 0.05), but the Dio3 and Sepx1 mRNA levels did not change. The results indicated that Se deficiency resulted in kidney dysfunction, activation of the NF-κB pathway, and a change in selenoprotein gene expression. The changes of inflammatory factor and selenoprotein gene expression levels were directly related to the abnormal renal functions induced by Se deficiency.

Activation of the mouse Oct4 promoter in medaka embryonic stem cells and its use for ablation of spontaneous differentiation.

PubMed

Hong, Yunhan; Winkler, Christoph; Liu, Tongming; Chai, Guixuan; Schartl, Manfred

2004-07-01

The determination and maintenance of the cell fate is ultimately due to differential gene activity. In the mouse, expression of the transcription factor Oct4 is high in totipotent inner cell mass, germ cells and undifferentiated embryonic stem (ES) cells, but dramatically reduced or extinct upon differentiation. Here, we show that medaka blastula embryos and cells of the ES cell line MES1 are able to activate the Oct4 promoter. Ectopic expression of a fusion gene for beta-galactosidase and neomycin resistance from the Oct4 promoter conferred resistance to G418. G418 selection led to a homogeneous population of undifferentiated ES cells which were able to undergo induced or directed differentiation into various cell types including neuron-like cells and melanocytes. Furthermore, GFP-labeled GOF18geo-MES1 cells after differentiation ablation were able to contribute to a wide variety of organ systems derived from all the three germ layers. Most importantly, we show that drug ablation of differentiation on the basis of Oct4 promoter is a useful tool to improve ES cell cultivation and chimera formation: MES1 cells after differentiation ablation appeared to be better donors than the parental MES1 line, as the permissive number of input donor cells increases from 100 to 200, resulting in an enhanced degree of chimerism. Taken together, some transcription factors and cis-acting regulatory sequences controlling totipotency-specific gene expression appear to be conserved between mammals and fish, and medaka ES cells offer an in vitro system for characterizing the expression of totipotency-specific genes such as putative Oct4 homologs from fish.

Exogenous Oct-4 Inhibits Lens Transdifferentiation in the Newt Notophthalmus viridescens

PubMed Central

Bhavsar, Rital B.; Tsonis, Panagiotis A.

2014-01-01

From the cocktail of four factors that were able to induce pluripotent stem cells from differentiated cells, Oct-4, c-Myc, Sox-2 and Klf4, only Oct-4 was not expressed during regeneration in newts. To explore the possible action of this stemness factor we developed an assay where we introduced exogenous Oct-4 protein to an in vitro system for lens regeneration in newts. We found that exogenous Oct-4 inhibits differentiation of iris pigmented epithelial cells into lens cells and also regulates Sox-2 and Pax-6, both important players during lens development. Thus, presence of Oct-4 hinders transdifferentiation of iris cells. PMID:25019378

Peripheral mRNA expression of pluripotency markers in bipolar disorder and the effect of long-term lithium treatment.

PubMed

Ferensztajn-Rochowiak, Ewa; Tarnowski, Maciej; Samochowiec, Jerzy; Michalak, Michal; Ratajczak, Mariusz Z; Rybakowski, Janusz K

2016-10-01

The aim was to evaluate the peripheral mRNA expression of pluripotency master transcriptional factors such as octamer-binding transcription factor 4 (Oct4), sex-determining region Y-box 2 (Sox2) and homeobox protein Nanog, in patients with bipolar disorder (BD), and the effect of long-term lithium treatment. Fifteen BD patients (aged 53±7years) not treated with lithium, with duration of illness>10years, 15 BD patients (aged 55±6years) treated with lithium for 8-40 years (mean 16years) and 15 control subjects (aged 50±5years) were included. Assessment of the mRNA levels of pluripotency markers (Oct-4, Sox 2 and Nanog) was performed, using the Real-time quantitative reverse transcription PCR (RQ-PCR) procedure, and the number of CD34+ very small embryonic-like stem cells (VSELs) was measured by flow cytometric analysis. In those BD patients not treated with lithium the expression of all three pluripotency genes was significantly higher than that in the control subjects. Oct-4, Sox2 and Nanog also positively correlated with the number of CD34+ VSELs/[ul] in this group. In the lithium-treated patients the mRNA levels of Nanog were significantly higher than in the control individuals and correlated with the number and % of CD34+ VSELs. The overexpression of the pluripotency master transcriptional factors in patients with a long duration of BD not treated with lithium, may contribute to the pathogenesis of the illness and make them potential biological markers of BD. Long-term lithium treatment may attenuate these excessive regenerative processes, especially in relation to the transcription factors Oct-4 and Sox2. Copyright © 2016. Published by Elsevier Urban & Partner Sp. z o.o.

Oct4 Methylation-Mediated Silencing As an Epigenetic Barrier Preventing Müller Glia Dedifferentiation in a Murine Model of Retinal Injury.

PubMed

Reyes-Aguirre, Luis I; Lamas, Monica

2016-01-01

Müller glia (MG) is the most abundant glial type in the vertebrate retina. Among its many functions, it is capable of responding to injury by dedifferentiating, proliferating, and differentiating into every cell types lost to damage. This regenerative ability is notoriously absent in mammals. We have previously reported that cultured mammalian MG undergoes a partial dedifferentiation, but fails to fully acquire a progenitor phenotype and differentiate into neurons. This might be explained by a mnemonic mechanism comprised by epigenetic traits, such as DNA methylation. To achieve a better understanding of this epigenetic memory, we studied the expression of pluripotency-associated genes, such as Oct4, Nanog , and Lin28 , which have been reported as necessary for regeneration in fish, at early times after NMDA-induced retinal injury in a mouse experimental model. We found that although Oct4 is expressed rapidly after damage (4 hpi), it is silenced at 24 hpi. This correlates with a significant decrease in the DNA methyltransferase Dnmt3b expression, which returns to basal levels at 24 hpi. By MS-PCR, we observed a decrease in Oct4 methylation levels at 4 and 12 hpi, before returning to a fully methylated state at 24 hpi. To demonstrate that these changes are restricted to MG, we separated these cells using a GLAST antibody coupled with magnetic beads. Finally, intravitreous administration of the DNA-methyltransferase inhibitor SGI-1027 induced Oct4 expression at 24 hpi in MG. Our results suggest that mammalian MG injury-induced dedifferentiation could be restricted by DNA methylation, which rapidly silences Oct4 expression, preventing multipotency acquisition.

Oct4 Methylation-Mediated Silencing As an Epigenetic Barrier Preventing Müller Glia Dedifferentiation in a Murine Model of Retinal Injury

PubMed Central

Reyes-Aguirre, Luis I.; Lamas, Monica

2016-01-01

Müller glia (MG) is the most abundant glial type in the vertebrate retina. Among its many functions, it is capable of responding to injury by dedifferentiating, proliferating, and differentiating into every cell types lost to damage. This regenerative ability is notoriously absent in mammals. We have previously reported that cultured mammalian MG undergoes a partial dedifferentiation, but fails to fully acquire a progenitor phenotype and differentiate into neurons. This might be explained by a mnemonic mechanism comprised by epigenetic traits, such as DNA methylation. To achieve a better understanding of this epigenetic memory, we studied the expression of pluripotency-associated genes, such as Oct4, Nanog, and Lin28, which have been reported as necessary for regeneration in fish, at early times after NMDA-induced retinal injury in a mouse experimental model. We found that although Oct4 is expressed rapidly after damage (4 hpi), it is silenced at 24 hpi. This correlates with a significant decrease in the DNA methyltransferase Dnmt3b expression, which returns to basal levels at 24 hpi. By MS-PCR, we observed a decrease in Oct4 methylation levels at 4 and 12 hpi, before returning to a fully methylated state at 24 hpi. To demonstrate that these changes are restricted to MG, we separated these cells using a GLAST antibody coupled with magnetic beads. Finally, intravitreous administration of the DNA-methyltransferase inhibitor SGI-1027 induced Oct4 expression at 24 hpi in MG. Our results suggest that mammalian MG injury-induced dedifferentiation could be restricted by DNA methylation, which rapidly silences Oct4 expression, preventing multipotency acquisition. PMID:27895551

Inhibition of NF-κB prevents the acidic bile-induced oncogenic mRNA phenotype, in human hypopharyngeal cells

PubMed Central

Vageli, Dimitra P.; Doukas, Sotirios G.; Sasaki, Clarence T.

2018-01-01

Bile-containing gastro-duodenal reflux has been clinically considered an independent risk factor in hypopharyngeal carcinogenesis. We recently showed that the chronic effect of acidic bile, at pH 4.0, selectively induces NF-κB activation and accelerates the transcriptional levels of genes, linked to head and neck cancer, in normal hypopharyngeal epithelial cells. Here, we hypothesize that NF-κB inhibition is capable of preventing the acidic bile-induced and cancer-related mRNA phenotype, in treated normal human hypopharyngeal cells. In this setting we used BAY 11-7082, a specific and well documented pharmacologic inhibitor of NF-κB, and we observed that BAY 11-7082 effectively inhibits the acidic bile-induced gene expression profiling of the NF-κB signaling pathway (down-regulation of 72 out of 84 analyzed genes). NF-κB inhibition significantly prevents the acidic bile-induced transcriptional activation of NF-κB transcriptional factors, RELA (p65) and c-REL, as well as genes related to and commonly found in established HNSCC cell lines. These include anti-apoptotic bcl-2, oncogenic STAT3, EGFR, ∆Np63, TNF-α and WNT5A, as well as cytokines IL-1β and IL-6. Our findings are consistent with our hypothesis demonstrating that NF-κB inhibition effectively prevents the acidic bile-induced cancer-related mRNA phenotype in normal human hypopharyngeal epithelial cells supporting an understanding that NF-κB may be a critical link between acidic bile and early preneoplastic events in this setting. PMID:29464041

Protein arginine methyltransferase 7-mediated microRNA-221 repression maintains Oct4, Nanog, and Sox2 levels in mouse embryonic stem cells.

PubMed

Chen, Tsai-Yu; Lee, Sung-Hun; Dhar, Shilpa S; Lee, Min Gyu

2018-03-16

The stemness maintenance of embryonic stem cells (ESCs) requires pluripotency transcription factors, including Oct4, Nanog, and Sox2. We have previously reported that protein arginine methyltransferase 7 (PRMT7), an epigenetic modifier, is an essential pluripotency factor that maintains the stemness of mouse ESCs, at least in part, by down-regulating the expression of the anti-stemness microRNA (miRNA) miR-24-2. To gain greater insight into the molecular basis underlying PRMT7-mediated maintenance of mouse ESC stemness, we searched for new PRMT7-down-regulated anti-stemness miRNAs. Here, we show that miR-221 gene-encoded miR-221-3p and miR-221-5p are anti-stemness miRNAs whose expression levels in mouse ESCs are directly repressed by PRMT7. Notably, both miR-221-3p and miR-221-5p targeted the 3' untranslated regions of mRNA transcripts of the major pluripotency factors Oct4, Nanog, and Sox2 to antagonize mouse ESC stemness. Moreover, miR-221-5p silenced also the expression of its own transcriptional repressor PRMT7. Transfection of miR-221-3p and miR-221-5p mimics induced spontaneous differentiation of mouse ESCs. CRISPR-mediated deletion of the miR-221 gene, as well as specific antisense inhibitors of miR-221-3p and miR-221-5p, inhibited the spontaneous differentiation of PRMT7-depleted mouse ESCs. Taken together, these findings reveal that the PRMT7-mediated repression of miR-221-3p and miR-221-5p expression plays a critical role in maintaining mouse ESC stemness. Our results also establish miR-221-3p and miR-221-5p as anti-stemness miRNAs that target Oct4 , Nanog , and Sox2 mRNAs in mouse ESCs. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

VIH from the mud crab is specifically expressed in the eyestalk and potentially regulated by transactivator of Sox9/Oct4/Oct1.

PubMed

Liu, Chunyun; Jia, Xiwei; Zou, Zhihua; Wang, Xiaowei; Wang, Yilei; Zhang, Ziping

2018-01-01

Vitellogenesis-inhibiting hormone (VIH) is known to regulate ovarian maturation by suppressing the synthesis of vitellogenin (Vtg) in crustaceans, which belongs to a member of crustacean hyperglycemic hormone (CHH) family synthesized and secreted from the X-organ/sinus gland complex of eyestalks. In this study, the cDNA, genomic DNA (gDNA) and the 5'-upstream regulatory (promoter region) sequences of VIH gene were obtained by conventional PCR, genome walker and tail-PCR techniques according to our transcriptomic database of Scylla paramamosain. The full-length cDNA of SpVIH is 634bp including 105bp 5'UTR, 151bp 3'UTR and 378bp ORF that encodes a peptide of 125 amino acids. The full length gDNA of SpVIH is 790bp containing two exons and one intron. The 5'-flanking promoter regions of SpVIH we isolated are 3070bp from the translation initiation (ATG) and 2398bp from the predicted transcription initiation (A), which consists of putative core promoter region and multiple potential transcription factor binding sites. SpVIH was only expressed in eyestalk. The expression level of SpVIH in eyestalk of female crab decreased gradually along with the development of ovary. As there is not cell line of crabs available, we chose the mature transfection system HEK293FT cell lines to explore the mechanism of transcription regulation of SpVIH in crabs. Sequential deletion assays using luciferase reporter gene in HEK293FT cells revealed that the possible promoter activity regions (including positive and negative transcription factors binding sites simultaneously) presented between pSpVIH-4 and pSpVIH-6. In order to further identify the crucial transcription factors binding site in this region, the site-directed mutagenesis of Sox9/Oct4/Oct1 binding site of pSpVIH-4 was created. The results demonstrated that the transcriptional activity of pSpVIH-4△ decreased significantly (p<0.05). Thus, it is reasonable to deduce that the Sox9/Oct4/Oct1 may be the essential positive transcription

HoxB2, HoxB4 and Alx4 genes are downregulated in the cadmium-induced omphalocele in the chick model.

PubMed

Doi, Takashi; Puri, Prem; Bannigan, John; Thompson, Jennifer

2010-10-01

In the chick embryo, administration of cadmium (Cd) induces omphalocele phenotype. HoxB2 and HoxB4, expressed in cell types that contribute to ventral body wall (VBW) formation, act together to mediate proper closure of the VBW, involving a key downstream transcription factor, Alx4. HoxB2 and HoxB4 knockout mice display VBW defects with specific downregulation of Alx4 gene expression, while homozygous Alx4 knockouts show omphalocele phenotype. Although the earliest histological changes in the Cd chick model occur commencing at 4H post treatment, the exact timing and molecular mechanism by which Cd acts is still unclear. We hypothesized that HoxB2, HoxB4 and Alx4 genes are downregulated during the critical timing of very early embryogenesis in the Cd-induced omphalocele chick model. After 60H incubation, chick embryos were harvested at 1H, 4H and 8H after treatment with saline or Cd and divided into controls and Cd group (n = 24 for each group). RT-PCR was performed to investigate the gene expression of HoxB2, HoxB4 and Alx4 and statistically analyzed (significance was accepted at p < 0.05). Immunohistochemical staining was also performed to evaluate the protein expression/distribution of HoxB2, HoxB4 and Alx4 in the chick embryo. The expression levels of HoxB2, HoxB4 and Alx4 gene at 4H were significantly downregulated in the Cd group as compared to controls, whereas there were no significant differences at the other time points. Immunoreactivity of HoxB2, HoxB4 and Alx4 at 4H is also markedly decreased in the ectoderm and the dermomyotome in the Cd chick model as compared to controls. Downregulation of HoxB2, HoxB4 and Alx4 expression during the narrow window of early embryogenesis may cause omphalocele in the Cd chick model by interfering with molecular signaling required for proper VBW formation. Furthermore, these results support the concept that HoxB2, HoxB4 and Alx4 genes work together to mediate proper VBW formation.

Of Mice and Snakes: A Tail of Oct4.

PubMed

Shylo, Natalia A; Weatherbee, Scott D

2016-08-08

The vertebrate axial skeleton comprises regions of specialized vertebrae, which vary in length between lineages. Aires et al. (2016) uncover a key role for Oct4 in determining trunk length in mice. Additionally, a heterochronic shift in Oct4 expression may underlie the extreme elongation of the trunk in snakes. Copyright © 2016 Elsevier Inc. All rights reserved.

Melatonin decreases estrogen receptor binding to estrogen response elements sites on the OCT4 gene in human breast cancer stem cells

PubMed Central

Lopes, Juliana; Arnosti, David; Trosko, James E.; Tai, Mei-Hui; Zuccari, Debora

2016-01-01

Cancer stem cells (CSCs) pose a challenge in cancer treatment, as these cells can drive tumor growth and are resistant to chemotherapy. Melatonin exerts its oncostatic effects through the estrogen receptor (ER) pathway in cancer cells, however its action in CSCs is unclear. Here, we evaluated the effect of melatonin on the regulation of the transcription factor OCT4 (Octamer Binding 4) by estrogen receptor alpha (ERα) in breast cancer stem cells (BCSCs). The cells were grown as a cell suspension or as anchorage independent growth, for the mammospheres growth, representing the CSCs population and treated with 10 nM estrogen (E2) or 10 μM of the environmental estrogen Bisphenol A (BPA) and 1 mM of melatonin. At the end, the cell growth as well as OCT4 and ERα expression and the binding activity of ERα to the OCT4 was assessed. The increase in number and size of mammospheres induced by E2 or BPA was reduced by melatonin treatment. Furthermore, binding of the ERα to OCT4 was reduced, accompanied by a reduction of OCT4 and ERα expression. Thus, melatonin treatment is effective against proliferation of BCSCs in vitro and impacts the ER pathway, demonstrating its potential therapeutic use in breast cancer. PMID:27551335

Yeast eIF4B binds to the head of the 40S ribosomal subunit and promotes mRNA recruitment through its N-terminal and internal repeat domains.

PubMed

Walker, Sarah E; Zhou, Fujun; Mitchell, Sarah F; Larson, Victoria S; Valasek, Leos; Hinnebusch, Alan G; Lorsch, Jon R

2013-02-01

Eukaryotic translation initiation factor (eIF)4B stimulates recruitment of mRNA to the 43S ribosomal pre-initiation complex (PIC). Yeast eIF4B (yeIF4B), shown previously to bind single-stranded (ss) RNA, consists of an N-terminal domain (NTD), predicted to be unstructured in solution; an RNA-recognition motif (RRM); an unusual domain comprised of seven imperfect repeats of 26 amino acids; and a C-terminal domain. Although the mechanism of yeIF4B action has remained obscure, most models have suggested central roles for its RRM and ssRNA-binding activity. We have dissected the functions of yeIF4B's domains and show that the RRM and its ssRNA-binding activity are dispensable in vitro and in vivo. Instead, our data indicate that the 7-repeats and NTD are the most critical domains, which mediate binding of yeIF4B to the head of the 40S ribosomal subunit via interaction with Rps20. This interaction induces structural changes in the ribosome's mRNA entry channel that could facilitate mRNA loading. We also show that yeIF4B strongly promotes productive interaction of eIF4A with the 43S•mRNA PIC in a manner required for efficient mRNA recruitment.

VH mutant rabbits lacking the VH1a2 gene develop a2+ B cells in the appendix by gene conversion-like alteration of a rearranged VH4 gene.

PubMed

Sehgal, D; Mage, R G; Schiaffella, E

1998-02-01

We investigated the molecular basis for the appearance of V(H)a2 allotype-bearing B cells in mutant Alicia rabbits. The mutation arose in an a2 rabbit; mutants exhibit altered expression of V(H) genes because of a small deletion encompassing V(H)1a2, the 3'-most gene in the V(H) locus. The V(H)1 gene is the major source of V(H)a allotype because this gene is preferentially rearranged in normal rabbits. In young homozygous ali/ali animals, the levels of a2 molecules found in the serum increase with age. In adult ali/ali rabbits, 20 to 50% of serum Igs and B cells bear a2 allotypic determinants. Previous studies suggested that positive selection results in expansion of a2 allotype-bearing B cells in the appendix of young mutant ali/ali rabbits. We separated appendix cells from a 6-wk-old Alicia rabbit by FACS based on the expression of surface IgM and a2 allotype. The VDJ portion of the expressed Ig mRNA was amplified from the IgM+ a2+ and IgM+ a2- populations by reverse transcriptase-PCR. The cDNAs from both populations were cloned and sequenced. Analysis of these sequences suggested that, in a2+ B cells, the first D proximal functional gene in Alicia rabbits, V(H)4a2, rearranged and was altered further by a gene conversion-like mechanism. Upstream V(H) genes were identified as potential gene sequence donors; V(H)9 was found to be the most frequently used gene donor. Among the a2- B cells, y33 was the most frequently rearranged gene.

Yeast eIF4B binds to the head of the 40S ribosomal subunit and promotes mRNA recruitment through its N-terminal and internal repeat domains

PubMed Central

Walker, Sarah E.; Zhou, Fujun; Mitchell, Sarah F.; Larson, Victoria S.; Valasek, Leos; Hinnebusch, Alan G.; Lorsch, Jon R.

2013-01-01

Eukaryotic translation initiation factor (eIF)4B stimulates recruitment of mRNA to the 43S ribosomal pre-initiation complex (PIC). Yeast eIF4B (yeIF4B), shown previously to bind single-stranded (ss) RNA, consists of an N-terminal domain (NTD), predicted to be unstructured in solution; an RNA-recognition motif (RRM); an unusual domain comprised of seven imperfect repeats of 26 amino acids; and a C-terminal domain. Although the mechanism of yeIF4B action has remained obscure, most models have suggested central roles for its RRM and ssRNA-binding activity. We have dissected the functions of yeIF4B’s domains and show that the RRM and its ssRNA-binding activity are dispensable in vitro and in vivo. Instead, our data indicate that the 7-repeats and NTD are the most critical domains, which mediate binding of yeIF4B to the head of the 40S ribosomal subunit via interaction with Rps20. This interaction induces structural changes in the ribosome’s mRNA entry channel that could facilitate mRNA loading. We also show that yeIF4B strongly promotes productive interaction of eIF4A with the 43S•mRNA PIC in a manner required for efficient mRNA recruitment. PMID:23236192

Interference of a speB 5' untranslated region partial deletion with mRNA degradation in Streptococcus pyogenes.

PubMed

Chen, Z; Mashburn-Warren, L; Merritt, J; Federle, M J; Kreth, J

2017-10-01

The 5' untranslated region (5' UTR) of an mRNA molecule embeds important determinants that modify its stability and translation efficiency. In Streptococcus pyogenes, a strict human pathogen, a gene encoding a secreted protease (speB) has a large 5' UTR with unknown functions. Here we describe that a partial deletion of the speB 5' UTR caused a general accumulation of mRNA in the stationary phase, and that the mRNA accumulation was due to retarded mRNA degradation. The phenotype was observed in several M serotypes harboring the partial deletion of the speB 5' UTR. The phenotype was triggered by the production of the truncated speB 5' UTR, but not by the disruption of the intact speB 5' UTR. RNase Y, a major endoribonuclease, was previously shown to play a central role in bulk mRNA turnover in stationary phase. However, in contrast to our expectations, we observed a weaker interaction between the truncated speB 5' UTR and RNase Y compared with the wild-type, which suggests that other unidentified RNA degrading components are required for the pleiotropic effects observed from the speB UTR truncation. Our study demonstrates how S. pyogenes uses distinct mRNA degradation schemes in exponential and stationary growth phases. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Quiescent Oct4+ Neural Stem Cells (NSCs) Repopulate Ablated Glial Fibrillary Acidic Protein+ NSCs in the Adult Mouse Brain.

PubMed

Reeve, Rachel L; Yammine, Samantha Z; Morshead, Cindi M; van der Kooy, Derek

2017-09-01

Adult primitive neural stem cells (pNSCs) are a rare population of glial fibrillary acidic protein (GFAP) - Oct4 + cells in the mouse forebrain subependymal zone bordering the lateral ventricles that give rise to clonal neurospheres in leukemia inhibitory factor in vitro. pNSC neurospheres can be passaged to self-renew or give rise to GFAP + NSCs that form neurospheres in epidermal growth factor and fibroblast growth factor 2, which we collectively refer to as definitive NSCs (dNSCs). Label retention experiments using doxycycline-inducible histone-2B (H2B)-green fluorescent protein (GFP) mice and several chase periods of up to 1 year quantified the adult pNSC cell cycle time as 3-5 months. We hypothesized that while pNSCs are not very proliferative at baseline, they may exist as a reserve pool of NSCs in case of injury. To test this function of pNSCs, we obtained conditional Oct4 knockout mice, Oct4 fl/fl ;Sox1 Cre (Oct4 CKO ), which do not yield adult pNSC-derived neurospheres. When we ablated the progeny of pNSCs, namely all GFAP + dNSCs, in these Oct4 CKO mice, we found that dNSCs did not recover as they do in wild-type mice, suggesting that pNSCs are necessary for dNSC repopulation. Returning to the H2B-GFP mice, we observed that the cytosine β-d-arabinofuranoside ablation of proliferating cells including dNSCs-induced quiescent pNSCs to proliferate and significantly dilute their H2B-GFP label. In conclusion, we demonstrate that pNSCs are the most quiescent stem cells in the adult brain reported to date and that their lineage position upstream of GFAP + dNSCs allows them to repopulate a depleted neural lineage. Stem Cells 2017;35:2071-2082. © 2017 AlphaMed Press.

High OCT4A levels drive tumorigenicity and metastatic potential of medulloblastoma cells

PubMed Central

Gonçalves da Silva, Patrícia Benites; Teixeira dos Santos, Márcia Cristina; Rodini, Carolina Oliveira; Kaid, Carolini; Leite Pereira, Márcia Cristina; Furukawa, Gabriela; Gimenes da Cruz, Daniel Sanzio; Goldfeder, Mauricio Barbugiani; Reily Rocha, Clarissa Ribeiro; Rosenberg, Carla; Okamoto, Oswaldo Keith

2017-01-01

Medulloblastoma is a highly aggressive pediatric brain tumor, in which sporadic expression of the pluripotency factor OCT4 has been recently correlated with poor patient survival. However the contribution of specific OCT4 isoforms to tumor aggressiveness is still poorly understood. Here, we report that medulloblastoma cells stably overexpressing the OCT4A isoform displayed enhanced clonogenic, tumorsphere generation, and invasion capabilities. Moreover, in an orthotopic metastatic model of medulloblastoma, OCT4A overexpressing cells generated more developed, aggressive and infiltrative tumors, with tumor-bearing mice attaining advanced metastatic disease and shorter survival rates. Pro-oncogenic OCT4A effects were expression-level dependent and accompanied by distinct chromosomal aberrations. OCT4A overexpression in medulloblastoma cells also induced a marked differential expression of non-coding RNAs, including poorly characterized long non-coding RNAs and small nucleolar RNAs. Altogether, our findings support the relevance of pluripotency-related factors in the aggravation of medulloblastoma traits classically associated with poor clinical outcome, and underscore the prognostic and therapeutic value of OCT4A in this challenging type of pediatric brain cancer. PMID:28186969

High OCT4A levels drive tumorigenicity and metastatic potential of medulloblastoma cells.

PubMed

da Silva, Patrícia Benites Gonçalves; Teixeira Dos Santos, Márcia Cristina; Rodini, Carolina Oliveira; Kaid, Carolini; Pereira, Márcia Cristina Leite; Furukawa, Gabriela; da Cruz, Daniel Sanzio Gimenes; Goldfeder, Mauricio Barbugiani; Rocha, Clarissa Ribeiro Reily; Rosenberg, Carla; Okamoto, Oswaldo Keith

2017-03-21

Medulloblastoma is a highly aggressive pediatric brain tumor, in which sporadic expression of the pluripotency factor OCT4 has been recently correlated with poor patient survival. However the contribution of specific OCT4 isoforms to tumor aggressiveness is still poorly understood. Here, we report that medulloblastoma cells stably overexpressing the OCT4A isoform displayed enhanced clonogenic, tumorsphere generation, and invasion capabilities. Moreover, in an orthotopic metastatic model of medulloblastoma, OCT4A overexpressing cells generated more developed, aggressive and infiltrative tumors, with tumor-bearing mice attaining advanced metastatic disease and shorter survival rates. Pro-oncogenic OCT4A effects were expression-level dependent and accompanied by distinct chromosomal aberrations. OCT4A overexpression in medulloblastoma cells also induced a marked differential expression of non-coding RNAs, including poorly characterized long non-coding RNAs and small nucleolar RNAs. Altogether, our findings support the relevance of pluripotency-related factors in the aggravation of medulloblastoma traits classically associated with poor clinical outcome, and underscore the prognostic and therapeutic value of OCT4A in this challenging type of pediatric brain cancer.

O-GlcNAc transferase regulates transcriptional activity of human Oct4.

PubMed

Constable, Sandii; Lim, Jae-Min; Vaidyanathan, Krithika; Wells, Lance

2017-10-01

O-linked β-N-acetylglucosamine (O-GlcNAc) is a single sugar modification found on many different classes of nuclear and cytoplasmic proteins. Addition of this modification, by the enzyme O-linked N-acetylglucosamine transferase (OGT), is dynamic and inducible. One major class of proteins modified by O-GlcNAc is transcription factors. O-GlcNAc regulates transcription factor properties through a variety of different mechanisms including localization, stability and transcriptional activation. Maintenance of embryonic stem (ES) cell pluripotency requires tight regulation of several key transcription factors, many of which are modified by O-GlcNAc. Octamer-binding protein 4 (Oct4) is one of the key transcription factors required for pluripotency of ES cells and more recently, the generation of induced pluripotent stem (iPS) cells. The action of Oct4 is modulated by the addition of several post-translational modifications, including O-GlcNAc. Previous studies in mice found a single site of O-GlcNAc addition responsible for transcriptional regulation. This study was designed to determine if this mechanism is conserved in humans. We mapped 10 novel sites of O-GlcNAc attachment on human Oct4, and confirmed a role for OGT in transcriptional activation of Oct4 at a site distinct from that found in mouse that allows distinction between different Oct4 target promoters. Additionally, we uncovered a potential new role for OGT that does not include its catalytic function. These results confirm that human Oct4 activity is being regulated by OGT by a mechanism that is distinct from mouse Oct4. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The HPV16 E7 oncoprotein increases the expression of Oct3/4 and stemness-related genes and augments cell self-renewal.

PubMed

Organista-Nava, Jorge; Gómez-Gómez, Yazmín; Ocadiz-Delgado, Rodolfo; García-Villa, Enrique; Bonilla-Delgado, José; Lagunas-Martínez, Alfredo; Tapia, Jesús Santa-Olalla; Lambert, Paul F; García-Carrancá, Alejandro; Gariglio, Patricio

2016-12-01

Oct3/4 is a transcription factor involved in maintenance of the pluripotency and self-renewal of stem cells. The E7 oncoprotein and 17β-estradiol (E 2 ) are key factors in cervical carcinogenesis. In the present study, we aimed to investigate the effect of the HPV16 E7 oncoprotein and E 2 on the expression pattern of Oct3/4, Sox2, Nanog and Fgf4. We also determined whether the E7 oncoprotein is associated with cell self-renewal. The results showed that Oct3/4, Sox2, Nanog and Fgf4 were upregulated by the E7 oncoprotein in vivo and in vitro and implicate E 2 in the upregulation of these factors in vivo. We also demonstrated that E7 is involved in cell self-renewal, suggesting that the HPV16 E7 oncoprotein upregulates Oct3/4, Sox2, Nanog and Fgf4 expression to maintain the self-renewal capacity of cancer stem cells. Copyright © 2016 Elsevier Inc. All rights reserved.

The POU Transcription Factor Oct-1 Represses Virus-Induced Interferon A Gene Expression

PubMed Central

Mesplède, Thibault; Island, Marie-Laure; Christeff, Nicolas; Petek, Fahrettin; Doly, Janine; Navarro, Sébastien

2005-01-01

Alpha interferon (IFN-α) and IFN-β are able to interfere with viral infection. They exert a vast array of biologic functions, including growth arrest, cell differentiation, and immune system regulation. This regulation extends from innate immunity to cellular and humoral adaptive immune responses. A strict control of expression is needed to prevent detrimental effects of unregulated IFN. Multiple IFN-A subtypes are coordinately induced in human and mouse cells infected by virus and exhibit differences in expression of their individual mRNAs. We demonstrated that the weakly expressed IFN-A11 gene is negatively regulated after viral infection, due to a distal negative regulatory element, binding homeoprotein pituitary homeobox 1 (Pitx1). Here we show that the POU protein Oct-1 binds in vitro and in vivo to the IFN-A11 promoter and represses IFN-A expression upon interferon regulatory factor overexpression. Furthermore, we show that Oct-1-deficient MEFs exhibit increased in vivo IFN-A gene expression and increased antiviral activity. Finally, the IFN-A expression pattern is modified in Oct-1-deficient MEFs. The broad representation of effective and potent octamer-like sequences within IFN-A promoters suggests an important role for Oct-1 in IFN-A regulation. PMID:16166650

Adenovirus-mediated interference of FABP4 regulates mRNA expression of ADIPOQ, LEP and LEPR in bovine adipocytes.

PubMed

Wei, S; Zan, L S; Wang, H B; Cheng, G; Du, M; Jiang, Z; Hausman, G J; McFarland, D C; Dodson, M V

2013-02-27

Fatty acid binding protein 4 (FABP4) is an important adipocyte gene, with roles in fatty acid transport and fat deposition in animals as well as human metabolic syndrome. However, little is known about the functional regulation of FABP4 at the cellular level in bovine. We designed and selected an effective shRNA (small hairpin RNA) against bovine FABP4, constructed a corresponding adenovirus (AD-FABP4), and then detected its influence on mRNA expression of four differentiation-related genes (PPAR(y), CEBPA, CEBPB, and SREBF1) and three lipid metabolism-related genes (ADIPOQ, LEP and LEPR) of adipocytes. The FABP4 mRNA content, derived from bovine adipocytes, decreased by 41% (P < 0.01) after 24 h and 66% (P < 0.01) after 72 h of AD-FABP4 infection. However, lower mRNA content of FABP4 did not significantly alter levels of differentiation-related gene expression at 24 h following AD-FABP4 treatment of bovine-derived preadipocytes (P = 0.54, 0.78, 0.89, and 0.94, respectively). Meanwhile, knocking down (partially silencing) FABP4 significantly decreased ADIPOQ (P < 0.05) and LEP (P < 0.01) gene expression after 24 h of AD-FABP4 treatment, decreased ADIPOQ (P < 0.01) and LEP (P < 0.01) gene expression, but increased LEPR mRNA expression (P < 0.01) after a 72-h treatment of bovine preadipocytes. We conclude that FABP4 plays a role in fat deposition and metabolic syndrome by regulating lipid metabolism-related genes (such as ADIPOQ, LEP and LEPR), without affecting the ability of preadipocytes to differentiate into adipocytes.

Denoising and 4D visualization of OCT images

PubMed Central

Gargesha, Madhusudhana; Jenkins, Michael W.; Rollins, Andrew M.; Wilson, David L.

2009-01-01

We are using Optical Coherence Tomography (OCT) to image structure and function of the developing embryonic heart in avian models. Fast OCT imaging produces very large 3D (2D + time) and 4D (3D volumes + time) data sets, which greatly challenge ones ability to visualize results. Noise in OCT images poses additional challenges. We created an algorithm with a quick, data set specific optimization for reduction of both shot and speckle noise and applied it to 3D visualization and image segmentation in OCT. When compared to baseline algorithms (median, Wiener, orthogonal wavelet, basic non-orthogonal wavelet), a panel of experts judged the new algorithm to give much improved volume renderings concerning both noise and 3D visualization. Specifically, the algorithm provided a better visualization of the myocardial and endocardial surfaces, and the interaction of the embryonic heart tube with surrounding tissue. Quantitative evaluation using an image quality figure of merit also indicated superiority of the new algorithm. Noise reduction aided semi-automatic 2D image segmentation, as quantitatively evaluated using a contour distance measure with respect to an expert segmented contour. In conclusion, the noise reduction algorithm should be quite useful for visualization and quantitative measurements (e.g., heart volume, stroke volume, contraction velocity, etc.) in OCT embryo images. With its semi-automatic, data set specific optimization, we believe that the algorithm can be applied to OCT images from other applications. PMID:18679509

Editing of the grapevine mitochondrial cytochrome b mRNA and molecular modeling of the protein.

PubMed

Islas-Osuna, María A; Silva-Moreno, Begonia; Caceres-Carrizosa, Nidia; García-Robles, Jesús M; Sotelo-Mundo, Rogerio R; Yepiz-Plascencia, Gloria M

2006-05-01

Cytochrome b (COB), the central catalytic subunit of ubiquinol cytochrome c reductase, is a component of the transmembrane electron transfer chain that generates proton motive force. Some plant COB mRNAs are processed by RNA editing, which changes the gene coding sequence. This report presents the sequences of the grapevine (Vitis vinifera L.) mitochondrial gene for apocytochrome b (cob), the edited mRNA and the deduced protein. Grapevine COB is 393 amino acids long and is 98% identical to homologs in rapeseed, Arabidopsis thaliana and Oenothera sp. Twenty-one C-U editing sites were identified in the grapevine cob mRNA, resulting in 20 amino acid changes. These changes increase the overall hydrophobicity of the protein and result in a more conserved protein. Molecular modeling of grapevine COB shows that residues changed by RNA editing fit the secondary structure characteristic of an integral membrane protein. This is the first complete mitochondrial gene reported for grapevine. Novel RNA editing sites were identified in grapevine cob, which have not been previously reported for other plants.

Biologically relevant effects of mRNA amplification on gene expression profiles.

PubMed

van Haaften, Rachel I M; Schroen, Blanche; Janssen, Ben J A; van Erk, Arie; Debets, Jacques J M; Smeets, Hubert J M; Smits, Jos F M; van den Wijngaard, Arthur; Pinto, Yigal M; Evelo, Chris T A

2006-04-11

Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. In the present study we compared microarray data obtained from amplified mRNA derived from biopsies of rat cardiac left ventricle and non-amplified mRNA derived from the same organ. Biopsies were linearly amplified to acquire enough material for a microarray experiment. Both amplified and unamplified samples were hybridized to the Rat Expression Set 230 Array of Affymetrix. Analysis of the microarray data showed that unamplified material of two different left ventricles had 99.6% identical gene expression. Gene expression patterns of two biopsies obtained from the same parental organ were 96.3% identical. Similarly, gene expression pattern of two biopsies from dissimilar organs were 92.8% identical to each other.Twenty-one percent of reporters called present in parental left ventricular tissue disappeared after amplification in the biopsies. Those reporters were predominantly seen in the low intensity range. Sequence analysis showed that reporters that disappeared after amplification had a GC-content of 53.7+/-4.0%, while reporters called present in biopsy- and whole LV-samples had an average GC content of 47.8+/-5.5% (P <0.001). Those reporters were also predicted to form significantly more (0.76+/-0.07 versus 0.38+/-0.1) and longer (9.4+/-0.3 versus 8.4+/-0.4) hairpins as compared to representative control reporters present before and after amplification. This study establishes that the gene expression profile obtained after amplification of mRNA of left ventricular

Biologically relevant effects of mRNA amplification on gene expression profiles

PubMed Central

van Haaften, Rachel IM; Schroen, Blanche; Janssen, Ben JA; van Erk, Arie; Debets, Jacques JM; Smeets, Hubert JM; Smits, Jos FM; van den Wijngaard, Arthur; Pinto, Yigal M; Evelo, Chris TA

2006-01-01

Background Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. In the present study we compared microarray data obtained from amplified mRNA derived from biopsies of rat cardiac left ventricle and non-amplified mRNA derived from the same organ. Biopsies were linearly amplified to acquire enough material for a microarray experiment. Both amplified and unamplified samples were hybridized to the Rat Expression Set 230 Array of Affymetrix. Results Analysis of the microarray data showed that unamplified material of two different left ventricles had 99.6% identical gene expression. Gene expression patterns of two biopsies obtained from the same parental organ were 96.3% identical. Similarly, gene expression pattern of two biopsies from dissimilar organs were 92.8% identical to each other. Twenty-one percent of reporters called present in parental left ventricular tissue disappeared after amplification in the biopsies. Those reporters were predominantly seen in the low intensity range. Sequence analysis showed that reporters that disappeared after amplification had a GC-content of 53.7+/-4.0%, while reporters called present in biopsy- and whole LV-samples had an average GC content of 47.8+/-5.5% (P <0.001). Those reporters were also predicted to form significantly more (0.76+/-0.07 versus 0.38+/-0.1) and longer (9.4+/-0.3 versus 8.4+/-0.4) hairpins as compared to representative control reporters present before and after amplification. Conclusion This study establishes that the gene expression profile obtained after

Molecular cloning and production of caprine recombinant Oct4 protein for generation induced pluripotent stem cells.

PubMed

Singhal, Dinesh K; Singhal, Raxita; Malik, Hruda N; Singh, Surender; Kumar, Sudarshan; Kaushik, Jai K; Mohanty, Ashok K; Malakar, Dhruba

2015-12-01

Oct4, pluripotency marker and transcription factor, expresses in embryonic stem cells. It plays a pivotal role in determination of stem cells fate. Up and down regulation of Oct4 causes differentiation of embryonic stem cells. It is one of the main transcription factors which remained concerned in every study related to induced pluripotent stem cell. Here, we report the production of goat Oct4 protein using plasmid and lentiviral based vectors. Firstly, Oct4 ORF was cloned in pAcGFP1-N1 plasmid vector and positive clones were screened with colony PCR. Oct4 was over-expressed in CHO-K1 cell line and expression was confirmed by observing green florescent protein expression in CHO-K1 cells. Secondly, Oct4 lentiviral expression construct has been prepared using pLenti-gw vector. Oct4 ORF was cloned into pLenti4/V5-DEST vector and viral particles were produced in 293FT cells. Oct4 viral particles were used to infect goat fibroblast cells. Oct4 expression was observed and confirmed in transfected goat fibroblast cells using RT-PCR. Detection of Oct4 protein in western blotting assay affirmed the capacity of over-expression of our Oct4 lentiviral vector. The lentiviral expression construct and recombinant Oct4 protein may be used for reprogramming of somatic cell into induced pluripotent stem cell.

BMP8B Is a Tumor Suppressor Gene Regulated by Histone Acetylation in Gastric Cancer.

PubMed

Wisnieski, Fernanda; Leal, Mariana Ferreira; Calcagno, Danielle Queiroz; Santos, Leonardo Caires; Gigek, Carolina Oliveira; Chen, Elizabeth Suchi; Artigiani, Ricardo; Demachki, Sâmia; Assumpção, Paulo Pimentel; Lourenço, Laércio Gomes; Burbano, Rommel Rodríguez; Smith, Marília Cardoso

2017-04-01

Different from genetic alterations, the reversible nature of epigenetic modifications provides an interesting opportunity for the development of clinically relevant therapeutics in different tumors. In this study, we aimed to screen and validate candidate genes regulated by the epigenetic marker associated with transcriptional activation, histone acetylation, in gastric cancer (GC). We first compared gene expression profile of trichostatin A-treated and control GC cell lines using microarray assay. Among the 55 differentially expressed genes identified in this analysis, we chose the up-regulated genes BMP8B and BAMBI for further analyses, that included mRNA and histone acetylation quantification in paired GC and nontumor tissue samples. BMP8B expression was reduced in GC compared to nontumor samples (P < 0.01). In addition, reduced BMP8B expression was associated with poorly differentiated GC (P = 0.02). No differences or histopathological associations were identified concerning BAMBI expression. Furthermore, acetylated H3K9 and H4K16 levels at BMP8B were increased in GC compared to nontumors (P < 0.05). However, reduced levels of acetylated H3K9 and H4K16 were associated with poorly differentiated GC (P < 0.05). Reduced levels of acetylated H3K9 was also associated with diffuse-type histological GC (P < 0.05). Notably, reduced BMP8B mRNA and acetylated H4K16 levels were positively correlated in poorly differentiated GC (P < 0.05). Our study demonstrated that BMP8B seems to be a tumor suppressor gene regulated by H4K16 acetylation in poorly differentiated GC. Therefore, BMP8B may be a potential target for TSA-based therapies in this GC sample subset. J. Cell. Biochem. 118: 869-877, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

[Gene copy number, mRNA transcription and protein expression of PD-1 gene in primary hepatocarcinoma patients].

PubMed

Fan, Hui-Min; Wu, Ling-Jie; Hu, Feng-Yu; Yang, Zhan

2012-08-01

To study the gene copy number, mRNA transcription and protien expression of programmed cell death 1 (PD-1) gene in primary hepatocellular carcinoma (PHC) patients and normal control individuals (NC) who are anti-HBs positive, and to investigate the variations in PD-1 gene copy numbers and its relationship with PHC. Real-time PCR was adopted to detect the PD-1 gene copy numbers and their mRNA expressions in peripheral blood mononuclear cells (PBMCs) from 24 samples of PHC patients and 26 of NC. Protein expression level of PD-1 on CD8+ T was analyzed by flow cytometry. In terms of number of PD-1 gene copy numbers, the percentage of cases of haploid (single) was 34.62% and 4.17% in PHC group and control group respectively while the percentage of cases of diploid (double) was 61.54% and 95.83% respectively. The difference between the two was statistically significant (chi2 = 7.639, P = 0.006). The rate of cases with double PD-1 gene copy numbers was found to be higher in patients with PHC than in control group. It was also found that the average expression of PD-1 mRNA was 2.35E-03 in control group and 1.23E-03 in PHC group. The expression level was significant lower in PHC group than that in control group when compared by using Mann-whitey technic (U = 153, P = 0.009). Furthermore, the frequency of PD-1 protein expression on CD8+ T cells was 3.72 +/- 0.32 in control group and 16.13 +/- 1.68 in PHC group. The level of PD-1 mRNA expression was higher in PHC and significant differences was shown between two groups (t = -7.073, P = 0.000). Our study suggests that the variation in PD-1 gene copy number may trigger primary hepatocellular carcinoma to HBV carriers. The relationship between the variation of PD-1 gene copy numbers and its association with primary hepatocellular carcinoma is worth further focus.

Oct4-GFP expression during transformation of gonocytes into spermatogonial stem cells in the perinatal mouse testis.

PubMed

Li, Ruili; Vannitamby, Amanda; Zhang, Jian-Guo; Fehmel, Emma L; Southwell, Bridget R; Hutson, John M

2015-12-01

In cryptorchidism perinatal failure to switch off Oct4, a germ cell (GC) marker, may lead to carcinoma in situ. We aimed to analyze Oct4 expression during mouse gonocyte transformation into spermatogonial stem cells (SSC). Testes from OG2 (Oct4-promoter driven eGFP) mice at embryonic day (E) 17 and postnatal day P0-10 underwent immunohistochemistry and immunoblotting. Antibodies against MVH, AMH, Ki67, and c-Kit were visualized by confocal microscopy. Numbers of Oct4-GFP(+) GC and Oct4-GFP(-) GC/tubule were counted using ImageJ. Data were analyzed using nonparametric one-way ANOVA. GC from E17-P4 were Oct4-GFP(+). Numbers of Oct4-GFP(-) GC/tubule increased from P6-10, whereas Oct4-GFP(+) GC/tubule numbers remained similar between P6 and P10. Sertoli cells proliferated from E17-P10, whereas GC only proliferated from P2. Gonocytes (Oct4-GFP(+)/c-Kit(-)) central in tubules migrated to the basement membrane to become prospermatogonia (Oct4-GFP(+)/c-Kit(-)) and then SSC (Oct4-GFP(+)/c-Kit(+)) from day 4 and further developed into Oct4-GFP(-)/c-Kit(+) at P6. In Oct4-GFP mice both centrally located gonocytes and prospermatogonia located at the tubular basement membrane were Oct4-GFP(+)/c-Kit(-) before further developing into SSC (Oct4-GFP(+)/c-Kit(+)). This indicates that Oct4 is important in gonocyte transformation into SSC. Understanding this process will aid GC tumor diagnostics and fertility potential in boys with UDT undergoing orchidopexy. Copyright © 2015 Elsevier Inc. All rights reserved.

Consequences of metaphase II oocyte cryopreservation on mRNA content.

PubMed

Chamayou, S; Bonaventura, G; Alecci, C; Tibullo, D; Di Raimondo, F; Guglielmino, A; Barcellona, M L

2011-04-01

We studied the consequences of freezing/thawing processes on mRNA contents in MII oocytes after slow-freezing/rapid thawing (SF/RT) and vitrification/warming (V/W) protocols, and compared the results to fresh MII oocytes. We quantified the nuclear transcript mRNA responsible for the translation of proteins belonging either to trans-regulatory protein family or to functional structural proteins such as proteins involved in DNA structural organization (NAP1L1, TOP1, H1F0H1), chromosomal structure maintenance (SMC, SCC3, RAD21, SMC1A, SMC1B, STAG3, REC8), mitochondrial energetic pathways (ATP5GJ, SDHC), cell cycle regulation and processes (CLTA, MAPK6, CKS2) and staminal cell potency-development competence stage (DPPA3, OCT4, FOXJ2). Surplus MII oocytes were donated from patients in IVF cycles and divided in three groups of 15 oocytes. Group 1 was comprised of non-cryopreserved oocytes and Groups 2 and 3 underwent SF/RT and V/W procedures, respectively. There was an overall decrease of mRNA extracted from cryopreserved oocytes compared to control group. Only 39.4% of mRNA content were preserved after SF/RT while 63.3% of mRNA content were maintained after V/W. Oocyte cryopreservation is associated with molecular injury associated with the decrease of stored mRNA. However the V/W protocol is more conservative than SF/RT resulting in a level of mRNA sufficient to maintain biologic functions in the subsequent fertilized oocyte. Copyright © 2011 Elsevier Inc. All rights reserved.

Differential expression of Oct4 variants and pseudogenes in normal urothelium and urothelial cancer.

PubMed

Wezel, Felix; Pearson, Joanna; Kirkwood, Lisa A; Southgate, Jennifer

2013-10-01

The transcription factor octamer-binding protein 4 (Oct4; encoded by POU5F1) has a key role in maintaining embryonic stem cell pluripotency during early embryonic development and it is required for generation of induced pluripotent stem cells. Controversy exists concerning Oct4 expression in somatic tissues, with reports that Oct4 is expressed in normal and in neoplastic urothelium carrying implications for a bladder cancer stem cell phenotype. Here, we show that the pluripotency-associated Oct4A transcript was absent from cultures of highly regenerative normal human urothelial cells and from low-grade to high-grade urothelial carcinoma cell lines, whereas alternatively spliced variants and transcribed pseudogenes were expressed in abundance. Immunolabeling and immunoblotting studies confirmed the absence of Oct4A in normal and neoplastic urothelial cells and tissues, but indicated the presence of alternative isoforms or potentially translated pseudogenes. The stable forced expression of Oct4A in normal human urothelial cells in vitro profoundly inhibited growth and affected morphology, but protein expression was rapidly down-regulated. Our findings demonstrate that pluripotency-associated isoform Oct4A is not expressed by normal or malignant human urothelium and therefore is unlikely to play a role in a cancer stem cell phenotype. However, our findings also indicate that urothelium expresses a variety of other Oct4 splice-variant isoforms and transcribed pseudogenes that warrant further study. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

A nontranscriptional role for Oct4 in the regulation of mitotic entry

PubMed Central

Zhao, Rui; Deibler, Richard W.; Lerou, Paul H.; Ballabeni, Andrea; Heffner, Garrett C.; Cahan, Patrick; Unternaehrer, Juli J.; Kirschner, Marc W.; Daley, George Q.

2014-01-01

Rapid progression through the cell cycle and a very short G1 phase are defining characteristics of embryonic stem cells. This distinct cell cycle is driven by a positive feedback loop involving Rb inactivation and reduced oscillations of cyclins and cyclin-dependent kinase (Cdk) activity. In this setting, we inquired how ES cells avoid the potentially deleterious consequences of premature mitotic entry. We found that the pluripotency transcription factor Oct4 (octamer-binding transcription factor 4) plays an unappreciated role in the ES cell cycle by forming a complex with cyclin–Cdk1 and inhibiting Cdk1 activation. Ectopic expression of Oct4 or a mutant lacking transcriptional activity recapitulated delayed mitotic entry in HeLa cells. Reduction of Oct4 levels in ES cells accelerated G2 progression, which led to increased chromosomal missegregation and apoptosis. Our data demonstrate an unexpected nontranscriptional function of Oct4 in the regulation of mitotic entry. PMID:25324523

Expression of Oct-4 is significantly associated with the development and prognosis of colorectal cancer

PubMed Central

ZHOU, HUAN; HU, YU; WANG, WEIPENG; MAO, YONG; ZHU, JINGJIE; ZHOU, BIN; SUN, JING; ZHANG, XUEGUANG

2015-01-01

Octamer-binding transcription factor 4 (Oct-4), is an essential transcription factor, which is required for pluripotency and self-renewal in embryonic stem cells and germ cells. It is also involved in maintaining cancer stem-like properties in certain types of tumor, and is an important biomarker for cancer stem cells. The present study investigated whether Oct-4 expression was associated with colorectal cancer (CRC). In order to achieve this, primary CRC tissues, matched non-tumor tissues and benign polyp tissues, representing different stages of carcinogenesis, were obtained, and Oct-4 expression was analyzed using reverse transcription-quantitative polymerase chain reaction, flow cytometry analysis and immunohistochemistry. Furthermore, the medical records of patients with CRC were reviewed, and clinicopathological analysis was performed in order to assess the association between Oct-4 expression and certain clinicopathological parameters. It was shown that the transcription and translation of Oct-4 increased in a stepwise manner, from non-tumor to benign polyp tissues, and from benign polyps to CRC tissues. Oct-4 expression in CRC was significantly correlated with histological grade (P=0.007), lymph node metastasis (P=0.027), distant metastasis (P=0.017) and TNM stage (P=0.041). Kaplan-Meier survival curve analysis demonstrated that Oct-4+ cases had a shorter median survival time (37.0 months) compared with Oct-4− cases (76.0 months; P=0.001). These results indicated that aberrant expression of Oct-4 may be involved in the development of CRC. Thus, Oct-4 may be a biomarker for the prediction, diagnosis or assessment of prognosis in CRC, in addition to a potential target for the treatment of this disease. PMID:26622555

Automated choroid segmentation of three-dimensional SD-OCT images by incorporating EDI-OCT images.

PubMed

Chen, Qiang; Niu, Sijie; Fang, Wangyi; Shuai, Yuanlu; Fan, Wen; Yuan, Songtao; Liu, Qinghuai

2018-05-01

The measurement of choroidal volume is more related with eye diseases than choroidal thickness, because the choroidal volume can reflect the diseases comprehensively. The purpose is to automatically segment choroid for three-dimensional (3D) spectral domain optical coherence tomography (SD-OCT) images. We present a novel choroid segmentation strategy for SD-OCT images by incorporating the enhanced depth imaging OCT (EDI-OCT) images. The down boundary of the choroid, namely choroid-sclera junction (CSJ), is almost invisible in SD-OCT images, while visible in EDI-OCT images. During the SD-OCT imaging, the EDI-OCT images can be generated for the same eye. Thus, we present an EDI-OCT-driven choroid segmentation method for SD-OCT images, where the choroid segmentation results of the EDI-OCT images are used to estimate the average choroidal thickness and to improve the construction of the CSJ feature space of the SD-OCT images. We also present a whole registration method between EDI-OCT and SD-OCT images based on retinal thickness and Bruch's Membrane (BM) position. The CSJ surface is obtained with a 3D graph search in the CSJ feature space. Experimental results with 768 images (6 cubes, 128 B-scan images for each cube) from 2 healthy persons, 2 age-related macular degeneration (AMD) and 2 diabetic retinopathy (DR) patients, and 210 B-scan images from other 8 healthy persons and 21 patients demonstrate that our method can achieve high segmentation accuracy. The mean choroid volume difference and overlap ratio for 6 cubes between our proposed method and outlines drawn by experts were -1.96µm3 and 88.56%, respectively. Our method is effective for the 3D choroid segmentation of SD-OCT images because the segmentation accuracy and stability are compared with the manual segmentation. Copyright © 2017. Published by Elsevier B.V.

Downregulation in GATA4 and Downstream Structural and Contractile Genes in the db/db Mouse Heart

PubMed Central

Broderick, Tom L.; Jankowski, Marek; Wang, Donghao; Danalache, Bogdan A.; Parrott, Cassandra R.; Gutkowska, Jolanta

2012-01-01

Reduced expression of GATA4, a transcriptional factor for structural and cardioprotective genes, has been proposed as a factor contributing to the development of cardiomyopathy. We investigated whether the reduction of cardiac GATA4 expression reported in diabetes alters the expression of downstream genes, namely, atrial natriuretic peptide (ANP), B-type natriuretic, peptide (BNP), and α- and β-myosin heavy chain (MHC). db/db mice, a model of type 2 diabetes, with lean littermates serving as controls, were studied. db/db mice exhibited obesity, hyperglycemia, and reduced protein expression of cardiac GLUT4 and IRAP (insulin-regulated aminopeptidase), the structural protein cosecreted with GLUT4. Hearts from db/db mice had reduced protein expression of GATA4 (~35%) with accompanying reductions in mRNA expression of ANP (~40%), BNP (~85%), and α-MHC mRNA (~50%) whereas expression of β-MHC mRNA was increased by ~60%. Low GATA4 was not explained by an increased ligase or atrogin1 expression. CHIP protein content was modestly downregulated (27%) in db/db mice whereas mRNA and protein expression of the CHIP cochaperone HSP70 was significantly decreased in db/db hearts. Our results indicate that low GATA4 in db/db mouse heart is accompanied by reduced expression of GATA4-regulated cardioprotective and structural genes, which may explain the development of cardiomyopathy in diabetes. PMID:22474596

CYP3A5 mRNA degradation by nonsense-mediated mRNA decay.

PubMed

Busi, Florent; Cresteil, Thierry

2005-09-01

The total CYP3A5 mRNA level is significantly greater in carriers of the CYP3A5*1 allele than in CYP3A5*3 homozygotes. Most of the CYP3A5*3 mRNA includes an intronic sequence (exon 3B) containing premature termination codons (PTCs) between exons 3 and 4. Two models were used to investigate the degradation of CYP3A5 mRNA: a CYP3A5 minigene consisting of CYP3A5 exons and introns 3 to 6 transfected into MCF7 cells, and the endogenous CYP3A5 gene expressed in HepG2 cells. The 3'-untranslated region g.31611C>T mutation has no effect on CYP3A5 mRNA decay. Splice variants containing exon 3B were more unstable than wild-type (wt) CYP3A5 mRNA. Cycloheximide prevents the recognition of PTCs by ribosomes: in transfected MCF7 and HepG2 cells, cycloheximide slowed down the degradation of exon 3B-containing splice variants, suggesting the participation of nonsense-mediated decay (NMD). When PTCs were removed from pseudoexon 3B or when UPF1 small interfering RNA was used to impair the NMD mechanism, the decay of the splice variant was reduced, confirming the involvement of NMD in the degradation of CYP3A5 splice variants. Induction could represent a source of variability for CYP3A5 expression and could modify the proportion of splice variants. The extent of CYP3A5 induction was investigated after exposure to barbiturates or steroids: CYP3A4 was markedly induced in a pediatric population compared with untreated neonates. However, no effect could be detected in either the total CYP3A5 RNA, the proportion of splice variant RNA, or the protein level. Therefore, in these carriers, induction is unlikely to switch on the phenotypic CYP3A5 expression in carriers of CYP3A5*3/*3.

Elevation of D4 dopamine receptor mRNA in postmortem schizophrenic brain.

PubMed

Stefanis, N C; Bresnick, J N; Kerwin, R W; Schofield, W N; McAllister, G

1998-01-01

The D4 dopamine (DA) receptor has been proposed to be a target for the development of a novel antipsychotic drug based on its pharmacological and distribution profile. There is much interest in whether D4 DA receptor levels are altered in schizophrenia, but the lack of an available receptor subtype-specific radioligand made this difficult to quantitate. In this study, we examined whether D4 mRNA levels are altered in different brain regions of schizophrenics compared to controls. Ribonuclease protection assays were carried out on total RNA samples isolated postmortem from frontal cortex and caudate brain regions of schizophrenics and matched controls. 32P-labelled RNA probes to the D4 DA receptor and to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), were hybridised with the RNA samples, digested with ribonucleases to remove unhybridised probe, and separated on 6% sequencing gels. Densitometer analysis on the subsequent autoradiogams was used to calculate the relative optical density of D4 mRNA compared to G3PDH mRNA. Statistical analysis of the data revealed a 3-fold higher level (P<0.011) of D4 mRNA in the frontal cortex of schizophrenics compared to controls. No increase was seen in caudate. D4 receptors could play a role in mediating dopaminergic activity in frontal cortex, an activity which may be malfunctioning in schizophrenia.

Specific knockdown of Oct4 and beta2-microglobulin expression by RNA interference in human embryonic stem cells and embryonic carcinoma cells.

PubMed

Matin, Maryam M; Walsh, James R; Gokhale, Paul J; Draper, Jonathan S; Bahrami, Ahmad R; Morton, Ian; Moore, Harry D; Andrews, Peter W

2004-01-01

We have used RNA interference (RNAi) to downregulate beta2-microglobulin and Oct4 in human embryonal carcinoma (hEC) cells and embryonic stem (hES) cells, demonstrating that RNAi is an effective tool for regulating specific gene activity in these human stem cells. The knockdown of Oct4 but not beta2-microglobulin expression in both EC and ES cells resulted in their differentiation, as indicated by a marked change in morphology, growth rate, and surface antigen phenotype, with respect to SSEA1, SSEA3, and TRA-1-60 expression. Expression of hCG and Gcm1 was also induced following knockdown of Oct4 expression, in both 2102Ep hEC cells and in H7 and H14 hES cells, consistent with the conclusion that, as in the mouse, Oct4 is required to maintain the undifferentiated stem cell state, and that differentiation to trophectoderm occurs in its absence. NTERA2 hEC cells also differentiated, but not to trophectoderm, suggesting their equivalence to a later stage of embryogenesis than other hEC and hES cells.

The Antagonistic Effect of Selenium on Cadmium-Induced Damage and mRNA Levels of Selenoprotein Genes and Inflammatory Factors in Chicken Kidney Tissue.

PubMed

Wang, Xinyue; Bao, Rongkun; Fu, Jing

2018-02-01

Selenium (Se) is a necessary trace mineral in the diet of humans and animals. Cadmium (Cd) is a toxic heavy metal that can damage animal organs, especially the kidneys. Antagonistic interactions between Se and Cd have been reported in previous studies. However, little is known about the effects of Se against Cd toxicity and on the mRNA levels of 25 selenoprotein genes and inflammatory factors in chicken kidneys. In the current study, we fed chickens with a Se-treated, Cd-treated, or Se/Cd treated diet for 90 days. We then analyzed the mRNA expression of inflammatory factors (including prostaglandin E synthase (PTGES), nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2)) and 25 selenoprotein genes (Gpx1, Gpx2, Gpx3, Gpx4, Txnrd1, Txnrd2, Txnrd3, Dio1, Dio2, Dio3, SPS2, Sepp1, SelPb, Sep15, Selh, Seli, Selm, Selo, Sels, Sepx1, Selu, Selk, Selw, Seln, Selt). The results demonstrated that Cd exposure increased the Cd content in the chicken kidneys, renal tubular epithelial cells underwent denaturation and necrosis, and the tubules became narrow or disappeared. However, Se supplementation reduced the Cd content in chicken kidneys and induced normal development of renal tubular epithelial cells. In addition, we also observed that Se alleviated the Cd-induced increase in the mRNA levels of inflammatory factors and ameliorated the Cd-induced downtrend in the mRNA levels of 25 selenoprotein genes in chicken kidneys.

Structure and expression of the rat CYP3A1 gene: isolation of the gene (P450/6betaB) and characterization of the recombinant protein.

PubMed

Nagata, K; Ogino, M; Shimada, M; Miyata, M; Gonzalez, F J; Yamazoe, Y

1999-02-15

A P450 gene (P450/6betaB) of the CYP3A subfamily was isolated from a rat genomic library. Nucleotide sequencing of the exons revealed a high similarity with P450PCN1 cDNA (Gonzalez et al. (1985), J. Biol. Chem. 260, 7345-7441), but differed in 41 nucleotides, resulting in 11 changes and 2 deletions of amino acid residues. The P450/6betaB spanned about 30 kbp and consisted of 13 exons, and was in exon number and size identical with CYP3A2 gene except in the 6th exon, which was shorter than that of CYP3A2. 6beta-B mRNA, which may be transcribed from P450/6betaB, was detected on Northern blotting and by reverse transcription-polymerase chain reaction (RT-PCR). Profiles of the developmental change and induction by a treatment with several chemicals were very similar to those of P450PCN1 mRNA reported previously. P450PCN1 mRNA and gene, however, were not detected by PCR in rats. To determine whether P450/6betaB encodes an active protein, a cDNA was isolated and expressed. Expression of 6beta-B cDNA in COS-1 cells was carried out and revealed that the recombinant protein comigrated with purified P4506beta-4 previously identified as CYP3A1. The recombinant 6beta-B protein showed similar turnover rate and regioselectivity for testosterone with purified P4506beta-4 by the simultaneous addition of NADPH-cytochrome P450 reductase and cytochrome b5. These data suggest that P450/6betaB encodes an active P450 form corresponding to CYP3A1 and P450PCN1 reported previously does not exist in rats. Copyright 1999 Academic Press.

β-glucuronidase use as a single internal control gene may confound analysis in FMR1 mRNA toxicity studies.

PubMed

Kraan, Claudine M; Cornish, Kim M; Bui, Quang M; Li, Xin; Slater, Howard R; Godler, David E

2018-01-01

Relationships between Fragile X Mental Retardation 1 (FMR1) mRNA levels in blood and intragenic FMR1 CGG triplet expansions support the pathogenic role of RNA gain of function toxicity in premutation (PM: 55-199 CGGs) related disorders. Real-time PCR (RT-PCR) studies reporting these findings normalised FMR1 mRNA level to a single internal control gene called β-glucuronidase (GUS). This study evaluated FMR1 mRNA-CGG correlations in 33 PM and 33 age- and IQ-matched control females using three normalisation strategies in peripheral blood mononuclear cells (PBMCs): (i) GUS as a single internal control; (ii) the mean of GUS, Eukaryotic Translation Initiation Factor 4A2 (EIF4A2) and succinate dehydrogenase complex flavoprotein subunit A (SDHA); and (iii) the mean of EIF4A2 and SDHA (with no contribution from GUS). GUS mRNA levels normalised to the mean of EIF4A2 and SDHA mRNA levels and EIF4A2/SDHA ratio were also evaluated. FMR1mRNA level normalised to the mean of EIF4A2 and SDHA mRNA levels, with no contribution from GUS, showed the most significant correlation with CGG size and the greatest difference between PM and control groups (p = 10-11). Only 15% of FMR1 mRNA PM results exceeded the maximum control value when normalised to GUS, compared with over 42% when normalised to the mean of EIF4A2 and SDHA mRNA levels. Neither GUS mRNA level normalised to the mean RNA levels of EIF4A2 and SDHA, nor to the EIF4A2/SDHA ratio were correlated with CGG size. However, greater variability in GUS mRNA levels were observed for both PM and control females across the full range of CGG repeat as compared to the EIF4A2/SDHA ratio. In conclusion, normalisation with multiple control genes, excluding GUS, can improve assessment of the biological significance of FMR1 mRNA-CGG size relationships.

A feedback loop comprising PRMT7 and miR-24-2 interplays with Oct4, Nanog, Klf4 and c-Myc to regulate stemness

PubMed Central

Lee, Sung-Hun; Chen, Tsai-Yu; Dhar, Shilpa S.; Gu, Bingnan; Chen, Kaifu; Kim, Young Zoon; Li, Wei; Lee, Min Gyu

2016-01-01

Self-renewal and pluripotency are two fundamental characteristics of embryonic stem cells (ESCs) and are controlled by diverse regulatory factors, including pluripotent factors, epigenetic regulators and microRNAs (miRNAs). Although histone methyltransferases are key epigenetic regulators, whether and how a histone methyltransferase forms a network with miRNAs and the core pluripotent factor system to regulate ESC stemness is little known. Here, we show that the protein arginine methyltransferase 7 (PRMT7) is a pluripotent factor essential for the stemness of mouse ESCs. PRMT7 repressed the miR-24-2 gene encoding miR-24-3p and miR-24-2-5p by upregulating the levels of symmetrically dimethylated H4R3. Notably, miR-24-3p targeted the 3′ untranslated regions (UTRs) of the major pluripotent factors Oct4, Nanog, Klf4 and c-Myc, whereas miR-24-2-5p silenced Klf4 and c-Myc expression. miR-24-3p and miR-24-2-5p also targeted the 3′UTR of their repressor gene Prmt7. miR-24-3p and miR-24-2-5p induced mouse ESC differentiation, and their anti-sense inhibitors substantially reversed spontaneous differentiation of PRMT7-depleted mouse ESCs. Oct4, Nanog, Klf4 and c-Myc positively regulated Prmt7 expression. These findings define miR-24-3p and miR-24-2-5p as new anti-pluripotent miRNAs and also reveal a novel epigenetic stemness-regulatory mechanism in which a double-negative feedback loop consisting of PRMT7 and miR-24-3p/miR24-2-5p interplays with Oct4, Nanog, Klf4 and c-Myc to control ESC stemness. PMID:27625395

A feedback loop comprising PRMT7 and miR-24-2 interplays with Oct4, Nanog, Klf4 and c-Myc to regulate stemness.

PubMed

Lee, Sung-Hun; Chen, Tsai-Yu; Dhar, Shilpa S; Gu, Bingnan; Chen, Kaifu; Kim, Young Zoon; Li, Wei; Lee, Min Gyu

2016-12-15

Self-renewal and pluripotency are two fundamental characteristics of embryonic stem cells (ESCs) and are controlled by diverse regulatory factors, including pluripotent factors, epigenetic regulators and microRNAs (miRNAs). Although histone methyltransferases are key epigenetic regulators, whether and how a histone methyltransferase forms a network with miRNAs and the core pluripotent factor system to regulate ESC stemness is little known. Here, we show that the protein arginine methyltransferase 7 (PRMT7) is a pluripotent factor essential for the stemness of mouse ESCs. PRMT7 repressed the miR-24-2 gene encoding miR-24-3p and miR-24-2-5p by upregulating the levels of symmetrically dimethylated H4R3. Notably, miR-24-3p targeted the 3' untranslated regions (UTRs) of the major pluripotent factors Oct4, Nanog, Klf4 and c-Myc, whereas miR-24-2-5p silenced Klf4 and c-Myc expression. miR-24-3p and miR-24-2-5p also targeted the 3'UTR of their repressor gene Prmt7 miR-24-3p and miR-24-2-5p induced mouse ESC differentiation, and their anti-sense inhibitors substantially reversed spontaneous differentiation of PRMT7-depleted mouse ESCs. Oct4, Nanog, Klf4 and c-Myc positively regulated Prmt7 expression. These findings define miR-24-3p and miR-24-2-5p as new anti-pluripotent miRNAs and also reveal a novel epigenetic stemness-regulatory mechanism in which a double-negative feedback loop consisting of PRMT7 and miR-24-3p/miR24-2-5p interplays with Oct4, Nanog, Klf4 and c-Myc to control ESC stemness. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Identification of stress responsive genes by studying specific relationships between mRNA and protein abundance.

PubMed

Morimoto, Shimpei; Yahara, Koji

2018-03-01

Protein expression is regulated by the production and degradation of mRNAs and proteins but the specifics of their relationship are controversial. Although technological advances have enabled genome-wide and time-series surveys of mRNA and protein abundance, recent studies have shown paradoxical results, with most statistical analyses being limited to linear correlation, or analysis of variance applied separately to mRNA and protein datasets. Here, using recently analyzed genome-wide time-series data, we have developed a statistical analysis framework for identifying which types of genes or biological gene groups have significant correlation between mRNA and protein abundance after accounting for potential time delays. Our framework stratifies all genes in terms of the extent of time delay, conducts gene clustering in each stratum, and performs a non-parametric statistical test of the correlation between mRNA and protein abundance in a gene cluster. Consequently, we revealed stronger correlations than previously reported between mRNA and protein abundance in two metabolic pathways. Moreover, we identified a pair of stress responsive genes ( ADC17 and KIN1 ) that showed a highly similar time series of mRNA and protein abundance. Furthermore, we confirmed robustness of the analysis framework by applying it to another genome-wide time-series data and identifying a cytoskeleton-related gene cluster (keratin 18, keratin 17, and mitotic spindle positioning) that shows similar correlation. The significant correlation and highly similar changes of mRNA and protein abundance suggests a concerted role of these genes in cellular stress response, which we consider provides an answer to the question of the specific relationships between mRNA and protein in a cell. In addition, our framework for studying the relationship between mRNAs and proteins in a cell will provide a basis for studying specific relationships between mRNA and protein abundance after accounting for potential

Heat shock instructs hESCs to exit from the self-renewal program through negative regulation of OCT4 by SAPK/JNK and HSF1 pathway.

PubMed

Byun, Kyunghee; Kim, Taek-Kyun; Oh, Jeehyun; Bayarsaikhan, Enkhjargal; Kim, Daesik; Lee, Min Young; Pack, Chan-Gi; Hwang, Daehee; Lee, Bonghee

2013-11-01

Environmental factors affect self-renewal of stem cells by modulating the components of self-renewal networks. Heat shock, an environmental factor, induces heat shock factors (HSFs), which up-regulate stress response-related genes. However, the link of heat shock to self-renewal of stem cells has not been elucidated yet. Here, we present the direct link of heat shock to a core stem cell regulator, OCT4, in the self-renewal network through SAPK/JNK and HSF1 pathway. We first showed that heat shock initiated differentiation of human embryonic stem cells (hESCs). Gene expression analysis revealed that heat shock increased the expression of many genes involved in cellular processes related to differentiation of stem cells. We then examined the effects of HSFs induced by heat shock on core self-renewal factors. Among HSFs, heat shock induced mainly HSF1 in hESCs. The HSF1 repressed the expression of OCT4, leading to the differentiation of hESCs and the above differentiation-related gene expression change. We further examined the effects of the upstream MAP (mitogen-activated protein) kinases of HSF1 on the repression of OCT4 expression by HSF1. Among the MAP kinases, SAPK/JNK controlled predominantly the repression of the OCT4 expression by HSF1. The direct link of heat shock to the core self-renewal regulator through SAPK/JNK and HSF1 provides a fundamental basis for understanding the effect of heat and other stresses involving activation of HSF1 on the self-renewal program and further controlling differentiation of hESCs in a broad spectrum of stem cell applications using these stresses. © 2013.

Micro-optical coherence tomography tracking of magnetic gene transfection via Au-Fe3O4 dumbbell nanoparticles

NASA Astrophysics Data System (ADS)

Shi, Wei; Liu, Xinyu; Wei, Chao; Xu, Zhichuan J.; Sim, Stanley Siong Wei; Liu, Linbo; Xu, Chenjie

2015-10-01

Heterogeneous Au-Fe3O4 dumbbell nanoparticles (NPs) are composed of Au NPs and Fe3O4 NPs that bring in optical and magnetic properties respectively. This article reports the engineering of Au-Fe3O4 NPs as gene carriers for magnetic gene transfection as well as contrast agents for micro-optical coherence tomography (μOCT). As a proof-of-concept, Au-Fe3O4 NPs are used to deliver the green fluorescent protein to HEK 293T cells and their entrance into the cells is monitored through μOCT.Heterogeneous Au-Fe3O4 dumbbell nanoparticles (NPs) are composed of Au NPs and Fe3O4 NPs that bring in optical and magnetic properties respectively. This article reports the engineering of Au-Fe3O4 NPs as gene carriers for magnetic gene transfection as well as contrast agents for micro-optical coherence tomography (μOCT). As a proof-of-concept, Au-Fe3O4 NPs are used to deliver the green fluorescent protein to HEK 293T cells and their entrance into the cells is monitored through μOCT. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05459a

Low-level lasers alter mRNA levels from traditional reference genes used in breast cancer cells

NASA Astrophysics Data System (ADS)

Teixeira, A. F.; Canuto, K. S.; Rodrigues, J. A.; Fonseca, A. S.; Mencalha, A. L.

2017-07-01

Cancer is among the leading causes of mortality worldwide, increasing the importance of treatment development. Low-level lasers are used in several diseases, but some concerns remains on cancers. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a technique used to understand cellular behavior through quantification of mRNA levels. Output data from target genes are commonly relative to a reference that cannot vary according to treatment. This study evaluated reference genes levels from MDA-MB-231 cells exposed to red or infrared lasers at different fluences. Cultures were exposed to red and infrared lasers, incubated (4 h, 37 °C), total RNA was extracted and cDNA synthesis was performed to evaluate mRNA levels from ACTB, GUSB and TRFC genes by RT-qPCR. Specific amplification was verified by melting curves and agarose gel electrophoresis. RefFinder enabled data analysis by geNorm, NormFinder and BestKeeper. Specific amplifications were obtained and, although mRNA levels from ACTB, GUSB or TRFC genes presented no significant variation through traditional statistical analysis, Excel-based tools revealed that the use of these reference genes are dependent of laser characteristics. Our data showed that exposure to low-level red and infrared lasers at different fluences alter the mRNA levels from ACTB, GUSB and TRFC in MDA-MB-231 cells.

Expression of calmodulin mRNA in rat olfactory neuroepithelium.

PubMed

Biffo, S; Goren, T; Khew-Goodall, Y S; Miara, J; Margolis, F L

1991-04-01

A calmodulin (CaM) cDNA was isolated by differential hybridization screening of a lambda gt10 library prepared from rat olfactory mucosa. This cDNA fragment, containing most of the open reading frame of the rat CaMI gene, was subcloned and used to characterize steady-state expression of CaM mRNA in rat olfactory neuroepithelium and bulb. Within the bulb mitral cells are the primary neuronal population expressing CaM mRNA. The major CaM mRNA expressed in the olfactory mucosa is 1.7 kb with smaller contributions from mRNAs of 4.0 and 1.4 kb. CaM mRNA was primarily associated with the olfactory neurons and, despite the cellular complexity of the tissue and the known involvement of CaM in diverse cellular processes, was only minimally evident in sustentacular cells, gland cells or respiratory epithelium. Following bulbectomy CaM mRNA declines in the olfactory neuroepithelium as does olfactory marker protein (OMP) mRNA. In contrast to the latter, CaM mRNA makes a partial recovery by one month after surgery. These results, coupled with those from in situ hybridization, indicate that CaM mRNA is expressed in both mature and immature olfactory neurons. The program regulating CaM gene expression in olfactory neurons is distinct from those controlling expression of B50/GAP43 in immature, or OMP in mature, neurons respectively.

Craniopharyngiomas express embryonic stem cell markers (SOX2, OCT4, KLF4, and SOX9) as pituitary stem cells but do not coexpress RET/GFRA3 receptors.

PubMed

Garcia-Lavandeira, Montserrat; Saez, Carmen; Diaz-Rodriguez, Esther; Perez-Romero, Sihara; Senra, Ana; Dieguez, Carlos; Japon, Miguel A; Alvarez, Clara V

2012-01-01

Adult stem cells maintain some markers expressed by embryonic stem cells and express other specific markers depending on the organ where they reside. Recently, stem/progenitor cells in the rodent and human pituitary have been characterized as expressing GFRA2/RET, PROP1, and stem cell markers such as SOX2 and OCT4 (GPS cells). Our objective was to detect other specific markers of the pituitary stem cells and to investigate whether craniopharyngiomas (CRF), a tumor potentially derived from Rathke's pouch remnants, express similar markers as normal pituitary stem cells. We conducted mRNA and Western blot studies in pituitary extracts, and immunohistochemistry and immunofluorescence on sections from normal rat and human pituitaries and 20 CRF (18 adamantinomatous and two papillary). Normal pituitary GPS stem cells localized in the marginal zone (MZ) express three key embryonic stem cell markers, SOX2, OCT4, and KLF4, in addition to SOX9 and PROP1 and β-catenin overexpression. They express the RET receptor and its GFRA2 coreceptor but also express the coreceptor GFRA3 that could be detected in the MZ of paraffin pituitary sections. CRF maintain the expression of SOX2, OCT4, KLF4, SOX9, and β-catenin. However, RET and GFRA3 expression was altered in CRF. In 25% (five of 20), both RET and GFRA3 were detected but not colocalized in the same cells. The other 75% (15 of 20) lose the expression of RET, GFRA3, or both proteins simultaneously. Human pituitary adult stem/progenitor cells (GPS) located in the MZ are characterized by expression of embryonic stem cell markers SOX2, OCT4, and KLF4 plus the specific pituitary embryonic factor PROP1 and the RET system. Redundancy in RET coreceptor expression (GFRA2 and GFRA3) suggest an important systematic function in their physiological behavior. CRF share the stem cell markers suggesting a common origin with GPS. However, the lack of expression of the RET/GFRA system could be related to the cell mislocation and deregulated

Nucleotide sequence determination of guinea-pig casein B mRNA reveals homology with bovine and rat alpha s1 caseins and conservation of the non-coding regions of the mRNA.

PubMed Central

Hall, L; Laird, J E; Craig, R K

1984-01-01

Nucleotide sequence analysis of cloned guinea-pig casein B cDNA sequences has identified two casein B variants related to the bovine and rat alpha s1 caseins. Amino acid homology was largely confined to the known bovine or predicted rat phosphorylation sites and within the 'signal' precursor sequence. Comparison of the deduced nucleotide sequence of the guinea-pig and rat alpha s1 casein mRNA species showed greater sequence conservation in the non-coding than in the coding regions, suggesting a functional and possibly regulatory role for the non-coding regions of casein mRNA. The results provide insight into the evolution of the casein genes, and raise questions as to the role of conserved nucleotide sequences within the non-coding regions of mRNA species. Images Fig. 1. PMID:6548375

Profiles of mRNA expression of related genes in the duck hypothalamus-pituitary growth axis during embryonic and early post-hatch development.

PubMed

Hu, Yan; Liu, Hongxiang; Song, Chi; Xu, Wenjuan; Ji, Gaige; Zhu, Chunhong; Shu, Jingting; Li, Huifang

2015-03-15

In this study, the ontogeny of body and liver weight and the pattern of related gene mRNA expression in the hypothalamus-pituitary growth axis (HPGA) of two different duck breeds (Anas platyrhynchos domestica) were compared during embryonic and post-hatch development. Duck hypothalamic growth hormone release hormone (GHRH), somatostatin (SS), pituitary growth hormone (GH), liver growth hormone receptor (GHR) and insulin-like growth factor-I (IGF-1) mRNA were first detected on the 13th embryonic day. During early duck development, SS maintained a lower expression status, whereas the other four genes exhibited highly significant variations in an age-specific manner. Highly significant breed specificity was observed with respect to hepatic IGF-1 mRNA expression, which showed a significant breed-age interaction effect. Compared with previous studies on chickens, significant species differences were observed regarding the mRNA expression of bird embryonic HPGA-related genes. During early development, highly significant breed and age specificity were observed with respect to developmental changes in body and liver weight, and varying degrees of significant linear correlation were found between these performances and the mRNA expression of HPGA-related genes in the duck HPGA. These results suggest that different genetic backgrounds may lead to differences in duck growth and HPGA-related gene mRNA expression, and the differential mRNA expression of related genes in the duck HPGA may be particularly important in the early growth of ducks. Furthermore, hepatic IGF-1 mRNA expression presented highly significant breed specificity, and evidence suggests the involvement of hepatic IGF-1 in mediating genetic effects on embryo and offspring growth in ducks. Copyright © 2015 Elsevier B.V. All rights reserved.

Effect of 3,3',5-triiodothyronine and 3,5-diiodothyronine on progesterone production, cAMP synthesis, and mRNA expression of STAR, CYP11A1, and HSD3B genes in granulosa layer of chicken preovulatory follicles.

PubMed

Sechman, A; Pawlowska, K; Hrabia, A

2011-10-01

In vitro studies were performed to assess whether stimulatory effects of triiodothyronine (T3) on progesterone (P4) production in a granulosa layer (GL) of chicken preovulatory follicles are associated with 3',5'-cyclic adenosine monophosphate (cAMP) synthesis and mRNA expression of STAR protein, CYP11A1, and HSD3B. Effects of 3,5-diiodothyronine (3,5-T2) on steroidogenic function in these follicles were also investigated. The GL of F3 to F1 follicles was incubated in medium supplemented with T3 or 3,5-T2, LH, or forskolin (F), and a combination of each iodothyronine with LH or F. Levels of P4 and cAMP in culture media were determined by RIA. Expression of genes involved in P4 synthesis (ie, STAR protein, CYP11A1, and HSD3B) in the GL of F3 to F1 follicles incubated in medium with T3 or 3,5-T2 and their combination with LH was performed by real-time PCR. Triiodothyronine increased basal and LH- and F-stimulated P4 secretion by preovulatory follicles. The 3,5-T2 elevated P4 synthesis by F3, had no effect on F2 follicles, and diminished P4 production by the GL of F1 follicles. It had no effect on LH-stimulated P4 production; however, it augmented F-stimulated P4 production by F2 and F1 follicles. Although T3 did not affect basal and F-stimulated cAMP synthesis by the GL of preovulatory follicles, it increased LH-stimulated synthesis of this nucleotide. However, 3,5-T2 elevated F-stimulated cAMP synthesis in F3 and F2 follicles; it did not change basal and LH-stimulated cAMP production. Triiodothyronine decreased basal STAR and CYP11A1 mRNAs in F3 follicles, increased them in F1 follicles, and elevated HSD3B mRNA levels in F1 follicles. Triiodothyronine augmented LH-stimulated STAR, CYP11A1, and HSD3B mRNA levels in F2 and CYP11A1 in F1 follicles. However, T3 decreased LH-stimulated STAR and HSD3B mRNA levels in F1 follicles. The 3,5-T2 did not affect basal STAR and CYP11A1 mRNA expression in all investigated follicles; however, it decreased LH-stimulated STAR

Rift Valley fever virus NSS gene expression correlates with a defect in nuclear mRNA export.

PubMed

Copeland, Anna Maria; Van Deusen, Nicole M; Schmaljohn, Connie S

2015-12-01

We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NSS gene, but not the N, GN or NSM genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NSS, confirming that expression of NSS is likely responsible for this phenomenon. Published by Elsevier Inc.

Phosphodiesterase 4 regulates the migration of B16-F10 melanoma cells.

PubMed

Watanabe, Yoshihiro; Murata, Taku; Shimizu, Kasumi; Morita, Hiroshi; Inui, Madoka; Tagawa, Toshiro

2012-08-01

Phosphodiesterases (PDEs) are important regulators of signal transduction processes. Eleven PDE gene families (PDE1-11) have been identified and several PDE isoforms are selectively expressed in various cell types. PDE4 family members specifically hydrolyze cyclic AMP (cAMP). Four genes (PDE4A-D) are known to encode PDE4 enzymes, with additional diversity generated by the use of alternative mRNA splicing and the use of different promoters. While PDE4 selective inhibitors show therapeutic potential for treating major diseases such as asthma and chronic obstructive pulmonary disease, little is known concerning the role of PDE4 in malignant melanoma. In this study, we examined the role of PDE4 in mouse B16-F10 melanoma cells. In these cells, PDE4 activity was found to be ∼60% of total PDE activity. RT-PCR detected only PDE4B and PDE4D mRNA. Cell growth was inhibited by the cAMP analog, 8-bromo-cAMP, but not by the specific PDE4 inhibitors, rolipram and denbufylline, which increased intracellular cAMP concentrations. Finally, migration of the B16-F10 cells was inhibited by the PDE4 inhibitors and 8-bromo-cAMP, while migration was increased by a protein kinase A (PKA) inhibitor, PKI(14-22), and was not affected by 8-pCPT-2'-O-Me-cAMP, which is an analog of exchange protein activated by cAMP (Epac). The inhibitory effect of rolipram on migration was reversed by PKI(14-22). Based on these results, PDE4 appears to play an important role in the migration of B16-F10 cells, and therefore may be a novel target for the treatment of malignant melanoma.

OCT/PS-OCT imaging of brachial plexus neurovascular structures

NASA Astrophysics Data System (ADS)

Raphael, David T.; Zhang, Jun; Zhang, Yaoping; Chen, Zhongping; Miller, Carol; Zhou, Li

2004-07-01

Introduction: Optical coherence tomography (OCT) allows high-resolution imaging (less than 10 microns) of tissue structures. A pilot study with OCT and polarization-sensitive OCT (PS-OCT) was undertaken to image ex-vivo neurovascular structures (vessels, nerves) of the canine brachial plexus. Methods: OCT is an interferometry-based optical analog of B-mode ultrasound, which can image through non-transparent biological tissues. With approval of the USC Animal Care and Use Committee, segments of the supra- and infraclavicular brachial plexus were excised from euthanized adult dogs, and the ex-vivo specimens were placed in cold pH-buffered physiologic solution. An OCT beam, in micrometer translational steps, scanned the fixed-position bisected specimens in transverse and longitudinal views. Two-dimensional images were obtained from identified arteries and nerves, with specific sections of interest stained with hematoxylin-eosin for later imaging through a surgical microscope. Results: with the beam scan direction transverse to arteries, the resulting OCT images showed an identifiable arterial lumen and arterial wall tissue layers. By comparison, transverse beam OCT images of nerves revealed a multitude of smaller nerve bundles contained within larger circular-shaped fascicles. PS-OCT imaging was helpful in showing the characteristic birefringence exhibited by arrayed neural structures. Discussion: High-resolution OCT imaging may be useful in the optical identification of neurovascular structures during attempted regional nerve blockade. If incorporated into a needle-shaped catheter endoscope, such a technology could prevent intraneural and intravascular injections immediately prior to local anesthetic injection. The major limitation of OCT is that it can form a coherent image of tissue structures only to a depth of 1.5 - 2 mm.

Multi-functional regulation of 4E-BP gene expression by the Ccr4-Not complex.

PubMed

Okada, Hirokazu; Schittenhelm, Ralf B; Straessle, Anna; Hafen, Ernst

2015-01-01

The mechanistic target of rapamycin (mTOR) signaling pathway is highly conserved from yeast to humans. It senses various environmental cues to regulate cellular growth and homeostasis. Deregulation of the pathway has been implicated in many pathological conditions including cancer. Phosphorylation cascades through the pathway have been extensively studied but not much is known about the regulation of gene expression of the pathway components. Here, we report that the mRNA level of eukaryotic translation initiation factor (eIF) subunit 4E-binding protein (4E-BP) gene, one of the key mTOR signaling components, is regulated by the highly conserved Ccr4-Not complex. RNAi knockdown of Not1, a putative scaffold protein of this protein complex, increases the mRNA level of 4E-BP in Drosophila Kc cells. Examination of the gene expression mechanism using reporter swap constructs reveals that Not1 depletion increases reporter mRNAs with the 3'UTR of 4E-BP gene, but decreases the ones with the 4E-BP promoter region, suggesting that Ccr4-Not complex regulates both degradation and transcription of 4E-BP mRNA. These results indicate that the Ccr4-Not complex controls expression of a single gene at multiple levels and adjusts the magnitude of the total effect. Thus, our study reveals a novel regulatory mechanism of a key component of the mTOR signaling pathway at the level of gene expression.

Replenishment of RANTES mRNA expression in activated eosinophils fromatopic asthmatics

PubMed Central

Velazquez, J R; Lacy, P; Moqbel, R

2000-01-01

Eosinophils have been shown to express the gene encoding regulated upon activation, normal T‐cell expressed and secreted (RANTES), a potent eosinophilotactic chemokine. RANTES protein expression in eosinophils has previously been shown to be up‐regulated by a number of agonists, including complement‐dependent factors (C3b/iC3b) and interferon‐γ (IFN‐γ). We hypothesized that gene expression of RANTES is regulated in these cells by eosinophil‐specific agonists. We analysed RANTES mRNA expression by reverse‐transcription polymerase chain reaction (RT‐PCR) in human peripheral blood eosinophils obtained from mild atopic asthmatics following stimulation over time. In resting eosinophils, a low level of RANTES mRNA was found to be constitutively expressed in all the atopic donors tested in this study (n = 6). Following stimulation with C3b/iC3b (serum‐coated surfaces), eosinophils released measurable levels of RANTES, while sustained transcript expression was detected for up to 24 hr of stimulation. In contrast, IFN‐γ (5 ng/ml) transiently and significantly (P < 0·05, n = 3) depleted relative amounts of RANTES PCR product (compared with β2‐microglobulin) after 1–4 hr of stimulation. RANTES transcript was again detectable after 24 hr of IFN‐γ incubation, suggesting that the pool of RANTES mRNA had been replenished. Other eosinophil‐active cytokines, interleukin‐3 (IL‐3), IL‐4, IL‐5 and granulocyte–macrophage colony‐stimulating factor, did not appear to modulate RANTES mRNA expression after 1 hr of incubation. The effect of IFN‐γ on RANTES mRNA was reversed by cycloheximide, suggesting that IFN‐γ may act by increasing the rate of translation of RANTES mRNA. These findings indicate that IFN‐γ may induce a rapid and transient effect on the translation and replenishment of RANTES mRNA in eosinophils. This novel observation supports the notion that eosinophils have the potential to replenish their stored and released

Inhibition of Oct 3/4 mitigates the cardiac progenitor-derived myocardial repair in infarcted myocardium.

PubMed

Zhao, Yu Tina; Du, Jianfeng; Chen, Youfang; Tang, Yaoliang; Qin, Gangjian; Lv, Guorong; Zhuang, Shougang; Zhao, Ting C

2015-12-24

Recent evidence has demonstrated that cardiac progenitor cells play an essential role in the induction of angiomyogenesis in infarcted myocardium. We and others have shown that engraftment of c-kit(+) cardiac stem cells (CSCs) into infarcted hearts led to myocardium regeneration and neovascularization, which was associated with an improvement of ventricular function. The purpose of this study is aimed at investigating the functional role of transcription factor (TF) Oct3/4 in facilitating CSCs to promote myocardium regeneration and preserve cardiac performance in the post-MI heart. c-kit(+) CSCs were isolated from adult hearts and re-introduced into the infarcted myocardium in which the mouse MI model was created by permanent ligation of the left anterior descending artery (LAD). The Oct3/4 of CSCs was inhibited by transfection of Oct3/4 siRNA, and transfection of CSCs with control siRNA serves as control groups. Myocardial functions were evaluated by echocardiographic measurement. Histological analysis was employed to assess newly formed cardiogenesis, neovascularization, and cell proliferations. Terminal deoxynucleotidyltransferase (TdT) nick-end labeling (TUNEL) was carried out to assess apoptotic cardiomyocytes. Real time polymerase chain reaction and Western blot were carried out to evaluate the level of Oct 3/4 in CSCs. Two weeks after engraftment, CSCs increased ventricular functional recovery as shown by a serial echocardiographic measurement, which is concomitant with the suppression of cardiac hypertrophy and attenuation of myocardial interstitial fibrosis. Suppression of Oct 3/4 of CSCs abrogated functional improvements and mitigated the hypertrophic response and cardiac remodeling. Transplantation of c-kit(+) CSCs into MI hearts promoted cardiac regeneration and neovascularization, which were abolished with the knockdown of Oct3/4. Additionally, suppression of Oct3/4 abrogated myocyte proliferation in the CSC-engrafted myocardium. Our results indicate

Regulation of LPS-induced mRNA expression of pro-inflammatory cytokines via alteration of NF-κB activity in mouse peritoneal macrophages exposed to fluoride.

PubMed

Tian, Yuhu; Huo, Meijun; Li, Guangsheng; Li, Yanyan; Wang, Jundong

2016-10-01

F toxicity to immune system, especially to macrophage, has been studied a lot recently. Nuclear factor-kappa B (NF-κB), as a transcription factor, plays a central role in immune and inflammatory responses via the regulation of downstream gene expression. Recent studies indicated that fluoride effect on inflammatory cytokine secretion, however, the molecular mechanism was less understood. In our study, peritoneal macrophages (PMs) were divided several groups and were administrated sodium fluoride (NaF, 50, 100, 200, 400, 800 μM) and/or lipopolysaccharide (LPS, 30 ng/mg). The mRNA expression of p65, inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) in macrophages exposed to fluoride was determined by quantitative real-time RT-PCR respectively. The translocation of NF-κB from cytoplasm to nucleus, which in a way reflects NF-κB activity, was demonstrated by Immunofluorescence and ELISA. Our results showed that fluoride had a dose-dependent effect on NF-κB activity, which coincided with LPS-induced mRNA expression of its downstream genes, iNOS and IL-1β. Fluoride alone causes no effect on gene expression. However, the mRNA expression of TNF-α showed non-NF-κB-dependent manner. Therefore, we come to the conclusion that fluoride can regulate LPS-induced mRNA expression of iNOS and IL-1β via NF-κB pathway in mouse peritoneal macrophages. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effect of UGT2B10, UGT2B17, FMO3, and OCT2 Genetic Variation on Nicotine and Cotinine Pharmacokinetics and Smoking in African Americans

PubMed Central

Taghavi, Taraneh; St. Helen, Gideon; Benowitz, Neal L.; Tyndale, Rachel F.

2017-01-01

OBJECTIVES Nicotine metabolism rates differ greatly among individuals, even after controlling for variation in the major nicotine metabolizing enzyme, CYP2A6. In this study, the impact of genetic variation in alternative metabolic enzymes and transporters on nicotine and cotinine pharmacokinetics and smoking was investigated. METHODS We examined the impact of UGT2B10, UGT2B17, FMO3, NAT1, and OCT2 variation on pharmacokinetics and smoking (total nicotine equivalents and topography), before and after stratifying by CYP2A6 genotype in 60 African American smokers who received a simultaneous intravenous infusion of deuterium-labeled nicotine and cotinine. RESULTS Variants in UGT2B10 and UGT2B17 were associated with urinary glucuronidation ratios (glucuronide/free substrate). UGT2B10 rs116294140 was associated with significant alterations in cotinine and modest alterations in nicotine pharmacokinetics. These alterations, however, were not sufficient to change nicotine intake or topography. Neither UGT2B10 rs61750900, UGT2B17*2, FMO3 rs2266782, nor NAT1 rs13253389 altered nicotine or cotinine pharmacokinetics among all subjects (n=60); or among individuals with reduced CYP2A6 activity (n=23). The organic cation transporter OCT2 rs316019 significantly increased nicotine and cotinine Cmax (p=0.005, p=0.02, respectively) and decreased nicotine clearance (p=0.05). UGT2B10 rs116294140 had no significant impact on the plasma or urinary trans-3’-hydroxycotinine/cotinine ratio, commonly used as a biomarker of CYP2A6 activity. CONCLUSIONS We demonstrated that polymorphisms in genes other than CYP2A6 represent minor sources of variation in nicotine pharmacokinetics, insufficient to alter smoking in African Americans. The change in cotinine pharmacokinetics with UGT2B10 rs116294140 highlights the UGT2B10 gene as a source of variability in cotinine as a biomarker of tobacco exposure among African American smokers. PMID:28178031

Nuclear delivery of recombinant OCT4 by chitosan nanoparticles for transgene-free generation of protein-induced pluripotent stem cells.

PubMed

Tammam, Salma; Malak, Peter; Correa, Daphne; Rothfuss, Oliver; Azzazy, Hassan M E; Lamprecht, Alf; Schulze-Osthoff, Klaus

2016-06-21

Protein-based reprogramming of somatic cells is a non-genetic approach for the generation of induced pluripotent stem cells (iPSCs), whereby reprogramming factors, such as OCT4, SOX2, KLF4 and c-MYC, are delivered as functional proteins. The technique is considered safer than transgenic methods, but, unfortunately, most protein-based protocols provide very low reprogramming efficiencies. In this study, we developed exemplarily a nanoparticle (NP)-based delivery system for the reprogramming factor OCT4. To this end, we expressed human OCT4 in Sf9 insect cells using a baculoviral expression system. Recombinant OCT4 showed nuclear localization in Sf9 cells indicating proper protein folding. In comparison to soluble OCT4 protein, encapsulation of OCT4 in nuclear-targeted chitosan NPs strongly stabilized its DNA-binding activity even under cell culture conditions. OCT4-loaded NPs enabled cell treatment with high micromolar concentrations of OCT4 and successfully delivered active OCT4 into human fibroblasts. Chitosan NPs therefore provide a promising tool for the generation of transgene-free iPSCs.

A nonimprinted Prader-Willi Syndrome (PWS)-region gene regulates a different chromosomal domain in trans but the imprinted pws loci do not alter genome-wide mRNA levels.

PubMed

Stefan, Mihaela; Portis, Toni; Longnecker, Richard; Nicholls, Robert D

2005-05-01

Prader-Willi syndrome (PWS) is a complex neurobehavioral disorder that results from loss of function of 10 clustered, paternally expressed genes in a 1.5-Mb region of chromosome 15q11-q13. Many of the primary PWS region genes appear to have nuclear RNA regulatory functions, suggesting that multiple genetic pathways could be secondarily affected in PWS. Using a transgenic mouse model of PWS (TgPWS) with an approximately 4-Mb chromosome 7C deletion of paternal origin that models the neonatal phenotype of the human syndrome we compared by oligonucleotide microarrays expression levels of approximately 12,000 genes and ESTs in TgPWS and wild-type brain. Hybridization data were processed with two distinct statistical algorithms and revealed a dramatically reduced expression of 4 imprinted genes within the deletion region in TgPWS mice, with 2 nonimprinted, codeleted genes reduced twofold. However, only 3 genes outside the deletion were significantly altered in TgPWS mouse brain, with approximately 1.5-fold up-regulation of mRNA levels. Remarkably, these genes map to a single chromosome domain (18B3), and by quantitative RT-PCR we show that 8 genes in this domain are up-regulated in TgPWS brain. These 18B3 genes were up-regulated in an equivalent manner in Angelman syndrome mouse (TgAS) brain, which has the same deletion but of maternal origin. Therefore, the trans-regulation of the chromosome 18B3 domain is due to decreased expression of a nonimprinted gene within the TgPWS/AS mouse deletion in mouse chromosome 7C. Most surprisingly, since 48-60% of the genome was screened, it appears that the imprinted mouse PWS loci do not widely regulate mRNA levels of other genes and may regulate RNA structure.

The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae.

PubMed

Huch, Susanne; Müller, Maren; Muppavarapu, Mridula; Gommlich, Jessie; Balagopal, Vidya; Nissan, Tracy

2016-10-15

The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC) reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs, or alternatively may imply a role for P bodies in mRNA stabilization. © 2016. Published by The Company of Biologists Ltd.

The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae

PubMed Central

Huch, Susanne; Müller, Maren; Muppavarapu, Mridula; Gommlich, Jessie; Balagopal, Vidya; Nissan, Tracy

2016-01-01

ABSTRACT The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC) reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs, or alternatively may imply a role for P bodies in mRNA stabilization. PMID:27543059

The P gene of bovine parainfluenza virus 3 expresses all three reading frames from a single mRNA editing site.

PubMed Central

Pelet, T; Curran, J; Kolakofsky, D

1991-01-01

The P gene of bovine parainfluenza virus 3 (bPIV3) contains two downstream overlapping ORFs, called V and D. By comparison with the mRNA editing sites of other paramyxoviruses, two editing sites were predicted for bPIV3; site a to express the D protein, and site b to express the V protein. Examination of the bPIV3 mRNAs, however, indicates that site b is non-functional whereas site a operates frequently. Insertions at site a give rise to both V and D protein mRNAs, because a very broad distribution of Gs is added when insertions occur. This broad distribution is very different from the editing sites of Sendai virus or SV5, where predominantly one form of edited mRNA containing either a one or two G insertion respectively is created, to access the single overlapping ORF of these viruses. A model is proposed to explain how paramyxoviruses control the range of G insertions on that fraction of the mRNAs where insertions occur. The bPIV3 P gene is unique as far as we know, in that a sizeable portion of the gene expresses all 3 reading frames as protein. bPIV3 apparently does this from a single editing site by removing the constraints which control the number of slippage rounds which take place. Images PMID:1846805

Alterations of cyclin-dependent kinase 4 inhibitor (p16INK4A/MTS1) gene structure and expression in acute lymphoblastic leukemias.

PubMed

Delmer, A; Tang, R; Senamaud-Beaufort, C; Paterlini, P; Brechot, C; Zittoun, R

1995-07-01

The cyclin-dependent kinase 4 (cdk4) inhibitor (p16INK4/MTS1/CDKN2) gene has been recently identified as a putative tumor suppressor gene because of the high frequency of homozygous deletion observed in numerous human tumor cell lines, including leukemias. However, results obtained from uncultured tumor samples have led to discussion of the relevance of these findings. Using reverse transcriptase polymerase chain reaction (RT-PCR) and Southern blot analysis, we have investigated p16INK4A gene at both RNA and genomic levels in various types of leukemias: acute myeloid leukemia (AML) (n = 23); acute lymphocytic leukemia (ALL) (n = 22) and B cell chronic lymphoproliferative disorders (CLPD) (n = 33). p16INK4A mRNA expression was not found in only 1/20 AML and 2/23 CLPD samples. Conversely, p16INK4A mRNA was not detected in 5/17 ALL cases, and intensity of PCR products were barely detectable in seven additional cases, possibly related to the contamination by normal cells in some cases. By Southern blotting, a homozygous deletion of p16INK4A gene was found in 6/17 ALL cases (35%) among which 4/6 were negative or weakly positive by RT-PCR assay. None of the five AML and 20 CLL samples studied had p16INK4A deletion. Sequence analysis of p16INK4A exon 2 did not show point mutation in two of these cases lacking mRNA expression. Our data provide further evidence that among hematological malignancies, ALL are the most likely to be associated with p16INK4A inactivation, mainly by homozygous gene deletion. Since most hematological malignancies-except ALL-are infrequently associated with p16INK4A and retinoblastoma (Rb) gene alteration it seems worthwhile to explore cdk4 and cdk6 expression to determine whether or not the disruption of the p16INK4A/Rb/cdk4/cdk6 regulatory loop might play a role in their pathogenesis.

Promoter methylation, mRNA expression of goat tumor‑associated genes and mRNA expression of DNA methyltransferase in enzootic nasal tumors.

PubMed

Quan, Zifang; Ye, Ni; Hao, Zhongxiang; Wen, Caifang; Liao, Hong; Zhang, Manli; Luo, Lu; Cao, Sanjie; Wen, Xintian; Wu, Rui; Yan, Qigui

2015-10-01

The aim of the present study was to investigate the promoter methylation status and mRNA expression of goat tumor‑associated genes, in addition to the mRNA expression of DNA methyltransferase genes in enzootic nasal tumors (ENT). Methylation‑specific polymerase chain reaction and SYBR Green reverse transcription‑quantitative polymerase chain reaction were used to detect the methylation status and the mRNA expression levels of DNA methyltransferases (DNMTs), O6‑methylguanine‑DNA methyltransferase (MGMT), the tumor suppressor genes P73, P53, GADD45G, CHFR and THBS1, the transcription factor CEBPA, the proto‑oncogenes KRAS, NRAS and C‑myc and EGFR in 24 nasal tumor tissue samples and 20 normal nasal epithelia tissue samples. The associations between promoter methylation and DNMT, and promoter methylation and mRNA expression of the genes were analyzed. The results indicated that the expression levels of DNMT1 increased by 56% compared with those in normal nasal epithelial tissues, while MGMT, DNMT3a and DNMT3b had similar expression levels in the two tissue types. The expression levels of P53 decreased by 36.8% and those of THBS1 by 43%, while C‑myc increased by 2.9‑fold and CEBPA by 2‑fold compared with that in normal nasal epithelial tissues. GADD45G, P73, CHFR and NRAS were observed to have similar expression levels in the two tissue types. However, no expression was observed for EGFR and KRAS. CHFR, GADD45G and THBS1 were identified to be methylated in tumor suppressor genes. The methylation expression rate of the CHFR gene was ~60% in the two tissue types and for THBS1 it was 100% in the nasal tumor tissues as opposed to 20% in the normal nasal epithelial tissues. The exhaustive methylation expression rate of GADD45G was 62.5% and the partial methylation expression rate was 37.5% in nasal tumor tissue, while no methylation was observed in normal nasal epithelial tissues. C‑myc was the only gene identified to be methylated amongst proto�

Enhanced levels of scrapie responsive gene mRNA in BSE-infected mouse brain.

PubMed

Dandoy-Dron, F; Benboudjema, L; Guillo, F; Jaegly, A; Jasmin, C; Dormont, D; Tovey, M G; Dron, M

2000-03-10

The expression of the mRNA of nine scrapie responsive genes was analyzed in the brains of FVB/N mice infected with bovine spongiform encephalopathy (BSE). The RNA transcripts of eight genes were overexpressed to a comparable extent in both BSE-infected and scrapie-infected mice, indicating a common series of pathogenic events in the two transmissible spongiform encephalopathies (TSEs). In contrast, the serine proteinase inhibitor spi 2, an analogue of the human alpha-1 antichymotrypsin gene, was overexpressed to a greater extent in the brains of scrapie-infected animals than in animals infected with BSE, reflecting either an agent specific or a mouse strain specific response. The levels of spi 2 mRNA were increased during the course of scrapie prior to the onset of clinical signs of the disease and the increase reached 11 to 45 fold relative to uninfected controls in terminally ill mice. Spi 2, in common with four of the other scrapie responsive genes studied, is known to be associated with pro-inflammatory processes. These observations underline the importance of cell reactivity in TSE. In addition, scrg2 mRNA the level of which is enhanced in TSE-infected mouse brain, was identified as a previously unrecognized long transcript of the murine aldolase C gene. However, the level of the principal aldolase C mRNA is unaffected in TSE. The increased representation of the longer transcript in the late stage of the disease may reflect changes in mRNA processing and/or stability in reactive astrocytes or in damaged Purkinje cells.

C4B gene influences intestinal microbiota through complement activation in patients with paediatric-onset inflammatory bowel disease.

PubMed

Nissilä, E; Korpela, K; Lokki, A I; Paakkanen, R; Jokiranta, S; de Vos, W M; Lokki, M-L; Kolho, K-L; Meri, S

2017-12-01

Complement C4 genes are linked to paediatric inflammatory bowel disease (PIBD), but the mechanisms have remained unclear. We examined the influence of C4B gene number on intestinal microbiota and in-vitro serum complement activation by intestinal microbes in PIBD patients. Complement C4A and C4B gene numbers were determined by genomic reverse transcription-polymerase chain reaction (RT-PCR) from 64 patients with PIBD (Crohn's disease or ulcerative colitis). The severity of the disease course was determined from faecal calprotectin levels. Intestinal microbiota was assessed using the HITChip microarray. Complement reactivity in patients was analysed by incubating their sera with Yersinia pseudotuberculosis and Akkermansia muciniphila and determining the levels of C3a and soluble terminal complement complex (SC5b-9) using enzyme immunoassays. The microbiota diversity was wider in patients with no C4B genes than in those with one or two C4B genes, irrespective of intestinal inflammation. C4B and total C4 gene numbers correlated positively with soluble terminal complement complex (TCC, SC5b-9) levels when patient serum samples were stimulated with bacteria. Our results suggest that the C4B gene number associates positively with inflammation in patients with PIBD. Multiple copies of the C4B gene may thus aggravate the IBD-associated dysbiosis through escalated complement reactivity towards the microbiota. © 2017 British Society for Immunology.

Gene regulation of atrial natriuretic peptide A, B, and C receptors in rat glomeruli.

PubMed

Itoh, K; Nonoguchi, H; Shiraishi, N; Tomita, K

1999-01-01

Atrial natriuretic peptide (ANP) has three types of receptor. We investigated the gene regulation of three types of ANP receptors (ANPR-A, B, and C) in rat glomeruli using reverse transcription coupled with competitive polymerase chain reaction (PCR). Competitive PCR revealed that ANPR-C mRNA expression was most abundant (ANPR-C > A > B) in glomeruli from control rats among mRNA expressions of three receptors, which were 20- to 15,000-fold higher than those in inner medullary collecting ducts. Two days' dehydration caused reversible decreases of ANPR-A, B, and C mRNAs by 50-80%. To determine the mechanisms of down-regulation of mRNA expression, isolated glomeruli were incubated in isotonic or hypertonic solution. Hyperosmolality induced by NaCl, mannitol or raffinose caused significant increases of ANPR-A, B, and C mRNA expression. Hypertonicity by urea showed smaller effects. ANP stimulated the expression of ANPR-A, B, and C mRNA in vitro. These results indicate that dehydration caused reversible decreases of ANPR-A, B, and C mRNA expression in glomeruli, and these decreases were not caused by increased plasma osmolality but probably by lower circulating levels of ANP.

Effects of PCB 126 and PCB 153 on secretion of steroid hormones and mRNA expression of steroidogenic genes (STAR, HSD3B, CYP19A1) and estrogen receptors (ERα, ERβ) in prehierarchical chicken ovarian follicles.

PubMed

Sechman, Andrzej; Batoryna, Marta; Antos, Piotr A; Hrabia, Anna

2016-12-15

The objective of this study was to assess the in vitro effects of dioxin-like PCB 126 and non-dioxin-like PCB 153 on basal and ovine LH (oLH)-stimulated testosterone (T) and estradiol (E2) secretion and expression of steroidogenic genes (STAR, HSD3B and CYP19A1) and estrogen receptors α (ERα) and β (ERβ) in white (WF) and yellowish (YF) prehierarchical follicles of the hen ovary. Steroid concentrations in a medium and gene expression in follicles following 6h of exposition were determined by RIA and real-time qPCR, respectively. Both PCBs increased basal and oLH-stimulated T secretion by the WF follicles. PCB 126 reduced basal E2 secretion by the WF follicles. PCB 153 elevated but PCB 126 reduced oLH-stimulated E2 secretion by the prehierarchical follicles. PCB 126 increased basal STAR and HSD3B and reduced CYP19A1 mRNA expression in these follicles. PCB 153 increased basal expression of STAR and HSD3B in YF follicles, but diminished HSD3B mRNA levels in the WF. The studied PCBs had an opposite effect on basal and oLH-stimulated CYP19A1 mRNA expression in prehierarchical follicles. Both PCBs modulated basal and inhibited oLH-stimulated ERα and ERβ gene expression in the prehierarchical follicles. In conclusion, data of the current study demonstrate the congener-specific effects of PCBs on sex steroid secretion by prehierarchical follicles of the chicken ovary, which are at least partly related to STAR, HSD3B and CYP19A1 gene expression. It is suggested that PCBs, by influencing follicular steroidogenesis and expression of estrogen receptors, may impair development and selection of yellowish follicles to the preovulatory hierarchy. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

[Carcinogenesis and its mechanism of mutant-type[12Asp]K-ras4B gene].

PubMed

Gui, Li-ming; Wei, Li-hui; Zhang, Ying-mei; Wang, Jian-liu; Wang, Ying; Chen, Ying; Ma, Da-long

2002-01-01

Ras gene plays an important role in the extra- and intra-cellular signal transduction pathway. It mediates series cascade reactions, and eventually actives transcriptional factors in nucleus. It is unknown on the mechanism of carcinogenesis of Ras gene in endometrial carcinoma, though K-ras mutant is very common in endometrial atypical hyperplasia and carcinoma. On basis of discovering the mutation in 12th codon of K-ras in endometrial carcinoma cell line, HEC-1A, we explored the carcinogenesis and molecular mechanism of mutant-type [12Asp] K-ras4B gene. (1) Full-length [12Asp]K-ras4B cDNA was amplified with RT-PCR, then inserted into pcDI eukaryotic expressive vector. (2) Morphological change, growth kinetics in vitro and tumorigencity in nude mice in vivo after-before transfection were observed. (3) To test the cell growth kinetics by methyl thiazolium tetrazolium (MTT) and [3H]thymidine incorporation method. (1) The authors have successfully constructed eukaryotic expression plasmid pcDI-[12Asp] K-ras4B; (2) To confirm that [12Asp] K-ras4B mutant can trigger the neoplastic transformation of NIH3T3 cells by test in vitro and in vivo. (3) After pMCV-RasN17 plasmid, a Ras mutant were transfected into pcDI-[12Asp] K-ras4B cells, the growth of this cell were restrained significantly in comparison with control group. (4) These findings indicate the expression of RafS621A resulted in remarkable inhibition in proliferation of pcDI-[12Asp]K-ras4B cell (P < 0.05). However, RafCAAX mutant can enhance pcDI-[12Asp]K-ras4B cell growth (P < 0.05). (1) [12Asp]K-ras4B gene alone is able to cause neoplastic transformation in NIH3T3 cells in vitro and in vivo. (2) [12Asp]K-ras4B-induced NIH3T3 cells neoplastic transformation required Raf signaling pathway.

SAC3B, a central component of the mRNA export complex TREX-2, is required for prevention of epigenetic gene silencing in Arabidopsis

PubMed Central

Yang, Yu; La, Honggui; Tang, Kai; Miki, Daisuke; Yang, Lan; Wang, Bangshing; Duan, Cheng-Guo; Nie, Wenfeng; Wang, Xingang; Wang, Siwen; Pan, Yufeng; Tran, Elizabeth J.; An, Lizhe; Zhang, Huiming; Zhu, Jian-Kang

2017-01-01

Epigenetic regulation is important for organismal development and response to the environment. Alteration in epigenetic status has been known mostly from the perspective of enzymatic actions of DNA methylation and/or histone modifications. In a genetic screen for cellular factors involved in preventing epigenetic silencing, we isolated an Arabidopsis mutant defective in SAC3B, a component of the conserved TREX-2 complex that couples mRNA transcription with nuleo-cytoplasmic export. Arabidopsis SAC3B dysfunction causes gene silencing at transgenic and endogenous loci, accompanied by elevation in the repressive histone mark H3K9me2 and by reduction in RNA polymerase Pol II occupancy. SAC3B dysfunction does not alter promoter DNA methylation level of the transgene d35S::LUC, although the DNA demethylase ROS1 is also required for d35S::LUC anti-silencing. THP1 and NUA were identified as SAC3B-associated proteins whose mutations also caused d35S::LUC silencing. RNA-DNA hybrid exists at the repressed loci but is unrelated to gene suppression by the sac3b mutation. Genome-wide analyses demonstrated minor but clear involvement of SAC3B in regulating siRNAs and DNA methylation, particularly at a group of TAS and TAS-like loci. Together our results revealed not only a critical role of mRNA-export factors in transcriptional anti-silencing but also the contribution of SAC3B in shaping plant epigenetic landscapes. PMID:27672037

Stem cells with FGF4-bFGF fused gene enhances the expression of bFGF and improves myocardial repair in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Xiang-Qi; Chen, Liang-Long, E-mail: xhzlyx@126.com; Fan, Lin

Highlights: • BFGF exists only in the cytoplasm of live cells. • BFGF cannot be secreted into the extracellular space to promote cell growth. • We combine the secretion-promoting signal peptide of FGF4. • We successfully modified BMSCs with the fused genes of FGF4-bFGF. • We promoted the therapeutic effects of transplanted BMSCs in myocardial infarction. - Abstract: The aim of this study was to investigate whether the modification of bone marrow-derived mesenchymal stem cells (BMSCs) with the fused FGF4 (fibroblast growth factor 4)-bFGF (basic fibroblast growth factor) gene could improve the expression and secretion of BFGF, and increase themore » efficacies in repairing infarcted myocardium. We used In-Fusion technique to construct recombinant lentiviral vectors containing the individual gene of bFGF, enhanced green fluorescent protein (EGFP), or genes of FGF4-bFGF and EGFP, and then transfected these lentiviruses into rat BMSCs. We conducted an in vitro experiment to compare the secretion of bFGF in BMSCs infected by these lentiviruses and also examined their therapeutic effects in the treatment of myocardial infraction in a rodent study. Sixty rats were tested in the following five conditions: Group-SHAM received only sham operation as controls; Group-AMI received only injection of placebo PBS buffer; Group-BMSC, Group-bFGF and Group-FGF4-bFGF received implantation of BMSCs with empty lentivirus, bFGF lentivirus, and FGF4-bFGF lentivirus, respectively. Our results found out that the transplanted FGF4-bFGF BMSCs had the highest survival rate, and also the highest myocardial expression of bFGF and microvascular density as evidenced by Western blotting and immunohistochemistry, respectively. As compared to other groups, the Group-FGF4-BFGF rats had the lowest myocardial fibrotic fraction, and the highest left ventricular ejection fraction. These results suggest that the modification of BMSCs with the FGF4-bFGF fused gene can not only increase the

Prognostic impact of MYC protein expression in central nervous system diffuse large B-cell lymphoma: comparison with MYC rearrangement and MYC mRNA expression.

PubMed

Son, Seung-Myoung; Ha, Sang-Yun; Yoo, Hae-Yong; Oh, Dongryul; Kim, Seok-Jin; Kim, Won-Seog; Ko, Young-Hyeh

2017-01-01

The prognostic role of MYC has been well documented in non-central nervous system diffuse large B-cell lymphoma; however, it remains controversial in central nervous system diffuse large B-cell lymphoma. To investigate the prognostic value of MYC, we analyzed the MYC protein expression by immunohistochemistry, mRNA expression by RNA in situ hybridization, and gene status by fluorescence in situ hybridization in 74 cases of central nervous system diffuse large B-cell lymphoma. Moreover, we examined the correlation between MYC translocation, mRNA expression, and protein expression. The mean percentage of MYC immunopositive cells was 49%. Using a 44% cutoff value, 49 (66%) cases showed MYC protein overexpression. The result of mRNA in situ hybridization using the RNA scope technology was obtained using the H-scoring system; the median value was 34.2. Using the cutoff value of 63.5, 16 (22%) cases showed MYC mRNA overexpression. MYC gene rearrangement was detected in five out of 68 (7%) cases. MYC translocation showed no statistically significant correlation with mRNA expression; however, all MYC translocation-positive cases showed MYC protein overexpression, with a higher mean percentage of MYC protein expression than that of translocation-negative cases (78 vs 48%, P=0.001). The level of MYC mRNA expression was moderately correlated with the level of MYC protein expression (P<0.001). The mean percentage of MYC protein expression in the high MYC mRNA group was higher than that in the low MYC mRNA group (70 vs 47%, P<0.001). A univariate analysis showed that age over 60 years, Eastern Cooperative Oncology Group (ECOG) performance status ≥2 and MYC protein overexpression were significantly associated with an increased risk of death. MYC translocation and MYC mRNA expression had no prognostic significance. On multivariate analysis, MYC protein overexpression and ECOG score retained prognostic significance.

Contribution of copy number variants involving nonsense-mediated mRNA decay pathway genes to neuro-developmental disorders.

PubMed

Nguyen, Lam S; Kim, Hyung-Goo; Rosenfeld, Jill A; Shen, Yiping; Gusella, James F; Lacassie, Yves; Layman, Lawrence C; Shaffer, Lisa G; Gécz, Jozef

2013-05-01

The nonsense-mediated mRNA decay (NMD) pathway functions not only to degrade transcripts containing premature termination codons (PTC), but also to regulate the transcriptome. UPF3B and RBM8A, important components of NMD, have been implicated in various forms of intellectual disability (ID) and Thrombocytopenia with Absent Radius (TAR) syndrome, which is also associated with ID. To gauge the contribution of other NMD factors to ID, we performed a comprehensive search for copy number variants (CNVs) of 18 NMD genes among individuals with ID and/or congenital anomalies. We identified 11 cases with heterozygous deletions of the genomic region encompassing UPF2, which encodes for a direct interacting protein of UPF3B. Using RNA-Seq, we showed that the genome-wide consequence of reduced expression of UPF2 is similar to that seen in patients with UPF3B mutations. Out of the 1009 genes found deregulated in patients with UPF2 deletions by at least 2-fold, majority (95%) were deregulated similarly in patients with UPF3B mutations. This supports the major role of deletion of UPF2 in ID. Furthermore, we found that four other NMD genes, UPF3A, SMG6, EIF4A3 and RNPS1 are frequently deleted and/or duplicated in the patients. We postulate that dosage imbalances of these NMD genes are likely to be the causes or act as predisposing factors for neuro-developmental disorders. Our findings further emphasize the importance of NMD pathway(s) in learning and memory.

Identification of Suitable Reference Genes for mRNA Studies in Bone Marrow in a Mouse Model of Hematopoietic Stem Cell Transplantation.

PubMed

Li, H; Chen, C; Yao, H; Li, X; Yang, N; Qiao, J; Xu, K; Zeng, L

2016-10-01

Bone marrow micro-environment changes during hematopoietic stem cell transplantation (HSCT) with subsequent alteration of genes expression. Quantitative polymerase chain reaction (q-PCR) is a reliable and reproducible technique for the analysis of gene expression. To obtain more accurate results, it is essential to find a reference during HSCT. However, which gene is suitable during HSCT remains unclear. This study aimed to identify suitable reference genes for mRNA studies in bone marrow after HSCT. C57BL/6 mice were treated with either total body irradiation (group T) or busulfan/cyclophosphamide (BU/CY) (group B) followed by infusion of bone marrow cells. Normal mice without treatments were served as a control. All samples (group T + group B + control) were defined as group G. On days 7, 14, and 21 after transplantation, transcription levels of 7 candidate genes, ACTB, B2M, GAPDH, HMBS, HPRT, SDHA, and YWHAZ, in bone marrow cells were measured by use of real-time quantitative PCR. The expression stability of these 7 candidate reference genes were analyzed by 2 statistical software programs, GeNorm and NormFinder. Our results showed that ACTB displayed the highest expression in group G, with lowest expression of PSDHA in group T and HPRT in groups B and G. Analysis of expression stability by use of GeNorm or NormFinder demonstrated that expression of B2M in bone marrow were much more stable during HSCT, compared with other candidate genes including commonly used reference genes GAPDH and ACTB. ACTB could be used as a suitable reference gene for mRNA studies in bone marrow after HSCT. Copyright © 2016 Elsevier Inc. All rights reserved.

Low-level lasers and mRNA levels of reference genes used in Escherichia coli

NASA Astrophysics Data System (ADS)

Teixeira, A. F.; Machado, Y. L. R. C.; Fonseca, A. S.; Mencalha, A. L.

2016-11-01

Low-level lasers are widely used for the treatment of diseases and antimicrobial photodynamic therapy. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is widely used to evaluate mRNA levels and output data from a target gene are commonly relative to a reference mRNA that cannot vary according to treatment. In this study, the level of reference genes from Escherichia coli exposed to red or infrared lasers at different fluences was evaluated. E. coli AB1157 cultures were exposed to red (660 nm) and infrared (808 nm) lasers, incubated (20 min, 37 °C), the total RNA was extracted, and cDNA synthesis was performed to evaluate mRNA levels from arcA, gyrA and rpoA genes by RT-qPCR. Melting curves and agarose gel electrophoresis were carried out to evaluate specific amplification. Data were analyzed by geNorm, NormFinder and BestKeeper. The melting curve and agarose gel electrophoresis showed specific amplification. Although mRNA levels from arcA, gyrA or rpoA genes presented no significant variations trough a traditional statistical analysis, Excel-based tools revealed that these reference genes are not suitable for E. coli cultures exposed to lasers. Our data showed that exposure to low-level red and infrared lasers at different fluences alter the mRNA levels from arcA, gyrA and rpoA in E. coli cells.

FPGA-based real-time swept-source OCT systems for B-scan live-streaming or volumetric imaging

NASA Astrophysics Data System (ADS)

Bandi, Vinzenz; Goette, Josef; Jacomet, Marcel; von Niederhäusern, Tim; Bachmann, Adrian H.; Duelk, Marcus

2013-03-01

We have developed a Swept-Source Optical Coherence Tomography (Ss-OCT) system with high-speed, real-time signal processing on a commercially available Data-Acquisition (DAQ) board with a Field-Programmable Gate Array (FPGA). The Ss-OCT system simultaneously acquires OCT and k-clock reference signals at 500MS/s. From the k-clock signal of each A-scan we extract a remap vector for the k-space linearization of the OCT signal. The linear but oversampled interpolation is followed by a 2048-point FFT, additional auxiliary computations, and a data transfer to a host computer for real-time, live-streaming of B-scan or volumetric C-scan OCT visualization. We achieve a 100 kHz A-scan rate by parallelization of our hardware algorithms, which run on standard and affordable, commercially available DAQ boards. Our main development tool for signal analysis as well as for hardware synthesis is MATLAB® with add-on toolboxes and 3rd-party tools.

NF-κB controls four genes encoding core enzymes of tricarboxylic acid cycle.

PubMed

Zhou, Fei; Xu, Xinhui; Wu, Jian; Wang, Danyang; Wang, Jinke

2017-07-20

NF-κB may promote tumor progression by altering cell metabolism. Hence, finding its target genes that are involved in cell metabolism is helpful for understanding its role in tumor growth. Here we discovered four metabolism-related target genes of this transcription factor. By analyzing a chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) data that characterizing the global binding sites (BSs) of NF-κB RelA in the TNFα-stimulated HeLa cells, we found that four genes that encode core enzymes of the tricarboxylic acid (TCA) cycle, including IDH1, IDH3A, ACO2, and SUCLA2, were multiply bound by this transcription factor. The subsequent bioinformatic analysis revealed that the NF-κB BSs contained many canonical κB sequences and the NF-κB-like DNA-binding motifs. Detection of ChIPed DNA with polymerase chain reaction (ChIP-PCR) also indicated that the NF-κB BSs were bound by NF-κB in both TNFα-treated HeLa and HepG2 cells. The reporter construct showed that the NF-κB BSs could activate the luciferase expression in cells in a NF-κB-specific manner. The quantitative PCR and Western blot detections demonstrated that NF-κB could regulate the expressions of IDH1, IDH3A, and ACO2 genes at both mRNA and protein levels and that of SUCLA2 gene at mRNA level in the TNFα-treated HeLa and HepG2 cells. Based on these investigations we identified the four genes as new target genes of NF-κB. The finding provides new insights into the role of NF-κB in cellular energetic metabolism, which may be beneficial for understanding the metabolic physiology of tumor growth. Copyright © 2017. Published by Elsevier B.V.

Analysis of IL12B Gene Variants in Inflammatory Bowel Disease

PubMed Central

Wagner, Johanna; Olszak, Torsten; Fries, Christoph; Tillack, Cornelia; Friedrich, Matthias; Beigel, Florian; Stallhofer, Johannes; Steib, Christian; Wetzke, Martin; Göke, Burkhard; Ochsenkühn, Thomas; Diegelmann, Julia; Czamara, Darina; Brand, Stephan

2012-01-01

Background IL12B encodes the p40 subunit of IL-12, which is also part of IL-23. Recent genome-wide association studies identified IL12B and IL23R as susceptibility genes for inflammatory bowel disease (IBD). However, the phenotypic effects and potential gene-gene interactions of IL12B variants are largely unknown. Methodology/Principal Findings We analyzed IL12B gene variants regarding association with Crohn's disease (CD) and ulcerative colitis (UC). Genomic DNA from 2196 individuals including 913 CD patients, 318 UC patients and 965 healthy, unrelated controls was analyzed for four SNPs in the IL12B gene region (rs3212227, rs17860508, rs10045431, rs6887695). Our analysis revealed an association of the IL12B SNP rs6887695 with susceptibility to IBD (p = 0.035; OR 1.15 [95% CI 1.01–1.31] including a trend for rs6887695 for association with CD (OR 1.41; [0.99–1.31], p = 0.066) and UC (OR 1.18 [0.97–1.43], p = 0.092). CD patients, who were homozygous C/C carriers of this SNP, had significantly more often non-stricturing, non-penetrating disease than carriers of the G allele (p = 6.8×10−5; OR = 2.84, 95% CI 1.66–4.84), while C/C homozygous UC patients had less often extensive colitis than G allele carriers (p = 0.029; OR = 0.36, 95% CI 0.14–0.92). In silico analysis predicted stronger binding of the minor C allele of rs6887695 to the transcription factor RORα which is involved in Th17 differentiation. Differences regarding the binding to the major and minor allele sequence of rs6887695 were also predicted for the transcription factors HSF1, HSF2, MZF1 and Oct-1. Epistasis analysis revealed weak epistasis of the IL12B SNP rs6887695 with several SNPs (rs11889341, rs7574865, rs7568275, rs8179673, rs10181656, rs7582694) in the STAT4 gene which encodes the major IL-12 downstream transcription factor STAT4 (p<0.05) but there was no epistasis between IL23R and IL12B variants. Conclusions/Significance The IL12B SNP rs6887695 modulates

DjhnRNPA2/B1-like gene is required for planarian regeneration and tissue homeostasis.

PubMed

Dong, Zimei; Yang, Tong; Yang, Yibo; Dou, He; Chen, Guangwen

2017-10-30

The hnRNPs play important roles in physiological processes in eukaryotic organisms by regulation of pre-mRNA after transcription, including pre-mRNA splicing, mRNA stability, DNA replication and repair and telomere maintenance and so on. However, it remains unclear about the specific functions of these genes. In this study, the full-length cDNA sequence of hnRNPA2/B1-like was first cloned from Dugesia japonica, and its roles were investigated by WISH and RNAi. The results showed that: (1) DjhnRNPA2/B1-like was highly conserved during animal evolution; (2) DjhnRNPA2/B1-like mRNA was mainly distributed each side of the body in intact worms and regenerative blastemas, and its expression levels were up-regulated on days 0 and 5 after amputation; (3) the intact and regenerating worms gradually lysed or lost regeneration capacity after DjhnRNPA2/B1-like RNAi; and (4) DjhnRNPA2/B1-like expression is induced by temperature and heavy metal ion stress. The data suggests that DjhnRNPA2/B1-like is a multiple functional gene, it plays important roles in regeneration and homeostatic maintenance and it is also involved in stress responses in planarians. Our work provides basic data for the study of regenerative mechanism and stress responses in freshwater planarians. Copyright © 2017. Published by Elsevier B.V.

A region rich in aspartic acid, arginine, tyrosine, and glycine (DRYG) mediates eukaryotic initiation factor 4B (eIF4B) self-association and interaction with eIF3.

PubMed Central

Méthot, N; Song, M S; Sonenberg, N

1996-01-01

The binding of mRNA to the ribosome is mediated by eukaryotic initiation factors eukaryotic initiation factor 4F (eIF4F), eIF4B, eIF4A, and eIF3, eIF4F binds to the mRNA cap structure and, in combination with eIF4B, is believed to unwind the secondary structure in the 5' untranslated region to facilitate ribosome binding. eIF3 associates with the 40S ribosomal subunit prior to mRNA binding. eIF4B copurifies with eIF3 and eIF4F through several purification steps, suggesting the involvement of a multisubunit complex during translation initiation. To understand the mechanism by which eIF4B promotes 40S ribosome binding to the mRNA, we studied its interactions with partner proteins by using a filter overlay (protein-protein [far Western]) assay and the two-hybrid system. In this report, we show that eIF4B self-associates and also interacts directly with the p170 subunit of eIF3. A region rich in aspartic acid, arginine, tyrosine, and glycine, termed the DRYG domain, is sufficient for self-association of eIF4B, both in vitro and in vivo, and for interaction with the p170 subunit of eIF3. These experiments suggest that eIF4B participates in mRNA-ribosome binding by acting as an intermediary between the mRNA and eIF3, via a direct interaction with the p170 subunit of eIF3. PMID:8816444

Efficient disruption of Zebrafish genes using a Gal4-containing gene trap

PubMed Central

2013-01-01

Background External development and optical transparency of embryos make zebrafish exceptionally suitable for in vivo insertional mutagenesis using fluorescent proteins to visualize expression patterns of mutated genes. Recently developed Gene Breaking Transposon (GBT) vectors greatly improve the fidelity and mutagenicity of transposon-based gene trap vectors. Results We constructed and tested a bipartite GBT vector with Gal4-VP16 as the primary gene trap reporter. Our vector also contains a UAS:eGFP cassette for direct detection of gene trap events by fluorescence. To confirm gene trap events, we generated a UAS:mRFP tester line. We screened 270 potential founders and established 41 gene trap lines. Three of our gene trap alleles display homozygous lethal phenotypes ranging from embryonic to late larval: nsf tpl6, atp1a3atpl10 and flrtpl19. Our gene trap cassette is flanked by direct loxP sites, which enabled us to successfully revert nsf tpl6, atp1a3atpl10 and flrtpl19 gene trap alleles by injection of Cre mRNA. The UAS:eGFP cassette is flanked by direct FRT sites. It can be readily removed by injection of Flp mRNA for use of our gene trap alleles with other tissue-specific GFP-marked lines. The Gal4-VP16 component of our vector provides two important advantages over other GBT vectors. The first is increased sensitivity, which enabled us to detect previously unnoticed expression of nsf in the pancreas. The second advantage is that all our gene trap lines, including integrations into non-essential genes, can be used as highly specific Gal4 drivers for expression of other transgenes under the control of Gal4 UAS. Conclusions The Gal4-containing bipartite Gene Breaking Transposon vector presented here retains high specificity for integrations into genes, high mutagenicity and revertibility by Cre. These features, together with utility as highly specific Gal4 drivers, make gene trap mutants presented here especially useful to the research community. PMID:24034702

Cell-cell contact regulates gene expression in CDK4-transformed mouse podocytes.

PubMed

Sakairi, Toru; Abe, Yoshifusa; Jat, Parmijit S; Kopp, Jeffrey B

2010-10-01

We transformed mouse podocytes by ectopic expression of cyclin-dependent kinase 4 (CDK4). Compared with podocytes transformed with a thermo-sensitive SV40 large T antigen mutant tsA58U19 (tsT podocytes), podocytes transformed with CDK4 (CDK4 podocytes) exhibited significantly higher expression of nephrin mRNA. Synaptopodin mRNA expression was significantly lower in CDK4 podocytes and in tsT podocytes under growth-permissive conditions (33°C) compared with tsT podocytes under growth-restricted conditions (37°C), which suggests a role for cell cycle arrest in synaptopodin mRNA expression. Confluent CDK4 podocytes showed significantly higher mRNA expression levels for nephrin, synaptopodin, Wilms tumor 1, podocalyxin, and P-cadherin compared with subconfluent cultures. We carried out experiments to clarify roles of various factors in the confluent podocyte cultures; our findings indicate that cell-cell contact promotes expression of five podocyte marker genes studied, that cellular quiescence increases synaptopodin and podocalyxin mRNA expression, and that soluble factors play a role in nephrin mRNA expression. Our findings suggest that CDK4 podocytes are useful tools to study podocyte biology. Furthermore, the role of cell-cell contact in podocyte gene expression may have relevance for podocyte function in vivo.

BORIS up-regulates OCT4 via histone methylation to promote cancer stem cell-like properties in human liver cancer cells.

PubMed

Liu, Qiuying; Chen, Kefei; Liu, Zhongjian; Huang, Yuan; Zhao, Rongce; Wei, Ling; Yu, Xiaoqin; He, Jingyang; Liu, Jun; Qi, Jianguo; Qin, Yang; Li, Bo

2017-09-10

Accumulating evidence has revealed the importance of cancer stem cells (CSCs) in chemoresistance and recurrence. BORIS, a testes-specific CTCF paralog, has been shown to be associated with stemness traits of embryonic cancer cells and epithelial CSCs. We previously reported that BORIS is correlated with the expression of the CSC marker CD90 in hepatocellular carcinoma (HCC). These results encourage us to wonder whether BORIS exerts functions on CSC-like traits of human liver cancer cells. Here, we report that BORIS was enriched in HCC tissues. Exogenous overexpression of BORIS promoted CSC-like properties, including self-renewal, chemoresistance, migration and invasion in Huh7 and HCCLM3 cells. Conversely, BORIS knockdown suppressed CSC-like properties in SMMC-7721 and HepG2 cells and inhibited tumorigenicity in SMMC-7721 cells. Moreover, BORIS alteration did not affect the DNA methylation status of the minimal promoter and exon 1 region of OCT4. However, BORIS overexpression enhanced the amount of BORIS bound on the OCT4 promoter and increased H3K4me2, while reducing H3K27me3; BORIS depletion decreased BORIS and H3K4me2 on the OCT4 promoter, while increasing H3K27me3. These results revealed that BORIS is associated with the CSC-like traits of human liver cancer cells through the epigenetic regulation of OCT4. Copyright © 2017 Elsevier B.V. All rights reserved.

Nogo-receptor gene activity: cellular localization and developmental regulation of mRNA in mice and humans.

PubMed

Josephson, Anna; Trifunovski, Alexandra; Widmer, Hans Ruedi; Widenfalk, Johan; Olson, Lars; Spenger, Christian

2002-11-18

Nogo (reticulon-4) is a myelin-associated protein that is expressed in three different splice variants, Nogo-A, Nogo-B, and Nogo-C. Nogo-A inhibits neurite regeneration in the central nervous system. Messenger RNA encoding Nogo is expressed in oligodendrocytes and central and peripheral neurons, but not in astrocytes or Schwann cells. Nogo is a transmembraneous protein; the extracellular domain is termed Nogo-66, and a Nogo-66-receptor (Nogo-R) has been identified. We performed in situ hybridization in human and mouse nervous tissues to map the cellular distribution of Nogo-R gene activity patterns in fetal and adult human spinal cord and sensory ganglia, adult human brain, and the nervous systems of developing and adult mice. In the human fetus Nogo-R was transcribed in the ventral horn of the spinal cord and in dorsal root ganglia. In adult human tissues Nogo-R gene activity was found in neocortex, hippocampus, amygdala, and a subset of large and medium-sized neurons of the dorsal root ganglia. Nogo-R mRNA was not expressed in the adult human spinal cord at detectable levels. In the fetal mouse, Nogo-R was diffusely expressed in brain, brainstem, trigeminal ganglion, spinal cord, and dorsal root ganglia at all stages. In the adult mouse strong Nogo-R mRNA expression was found in neurons in neocortex, hippocampus, amygdala, habenula, thalamic nuclei, brainstem, the granular cell layer of cerebellum, and the mitral cell layer of the olfactory bulb. Neurons in the adult mouse striatum, the medial septal nucleus, and spinal cord did not express Nogo-R mRNA at detectable levels. In summary, Nogo-66-R mRNA expression in humans and mice was observed in neurons of the developing nervous system Expression was downregulated in the adult spinal cord of both species, and specific expression patterns were seen in the adult brain. Copyright 2002 Wiley-Liss, Inc.

CD86 and beta2-adrenergic receptor signaling pathways, respectively, increase Oct-2 and OCA-B Expression and binding to the 3'-IgH enhancer in B cells.

PubMed

Podojil, Joseph R; Kin, Nicholas W; Sanders, Virginia M

2004-05-28

Stimulation of CD86 (formerly known as B7-2) and/or the beta2-adrenergic receptor on a CD40 ligand/interleukin-4-activated B cell increased the rate of mature IgG1 transcription. To identify the mechanism responsible for this effect, we determined whether CD86 and/or beta2-adrenergic receptor stimulation regulated transcription factor expression and binding to the 3'-IgH enhancer in vitro and in vivo. We showed that CD86 stimulation increased the nuclear localization of NF-kappaB1 (p50) and phosphorylated RelA (p65) and increased Oct-2 expression and binding to the 3'-IgH enhancer, in a protein kinase C-dependent manner. These effects were lost when CD86-deficient or NF-kappaB1-deficient B cells were used. CD86 stimulation also increased the level of IkappaB-alpha phosphorylation but in a protein kinase C-independent manner. Beta2-adrenergic receptor stimulation increased CREB phosphorylation, OCA-B expression, and OCA-B binding to the 3'-IgH enhancer in a protein kinase A-dependent manner, an effect lost when beta2-adrenergic receptor-deficient B cells were used. Also, the beta2-adrenergic receptor-induced increase in the level of mature IgG1 transcript was lost when OCA-B-deficient B cells were used. These data are the first to show that CD86 stimulation up-regulates the expression of the transcription factor Oct-2 in a protein kinase C- and NF-kappaB1-dependent manner, and that beta2-adrenergic receptor stimulation up-regulates the expression of the coactivator OCA-B in a protein kinase A-dependent manner to cooperate with Oct-2 binding to the 3'-IgH enhancer.

Quantification of cytokine mRNA in peripheral blood mononuclear cells using branched DNA (bDNA) technology.

PubMed

Shen, L P; Sheridan, P; Cao, W W; Dailey, P J; Salazar-Gonzalez, J F; Breen, E C; Fahey, J L; Urdea, M S; Kolberg, J A

1998-06-01

Changes in the patterns of cytokine expression are thought to be of central importance in human infectious and inflammatory diseases. As such, there is a need for precise, reproducible assays for quantification of cytokine mRNA that are amenable to routine use in a clinical setting. In this report, we describe the design and performance of a branched DNA (bDNA) assay for the direct quantification of multiple cytokine mRNA levels in peripheral blood mononuclear cells (PBMCs). Oligonucleotide target probe sets were designed for several human cytokines, including TNFalpha, IL-2, IL-4, IL-6, IL-10, and IFNgamma. The bDNA assay yielded highly reproducible quantification of cytokine mRNAs, exhibited a broad linear dynamic range of over 3-log10, and showed a sensitivity sufficient to measure at least 3000 molecules. The potential clinical utility of the bDNA assay was explored by measuring cytokine mRNA levels in PBMCs from healthy and immunocompromised individuals. Cytokine expression levels in PBMCs from healthy blood donors were found to remain relatively stable over a one-month period of time. Elevated levels of IFNgamma mRNA were detected in PBMCs from HIV-1 seropositive individuals, but no differences in mean levels of TNFalpha or IL-6 mRNA were detected between seropositive and seronegative individuals. By providing a reproducible method for quantification of low abundance transcripts in clinical specimens, the bDNA assay may be useful for studies addressing the role of cytokine expression in disease.

Accumulation of catechins in tea in relation to accumulation of mRNA from genes involved in catechin biosynthesis.

PubMed

Eungwanichayapant, P D; Popluechai, S

2009-02-01

Catechins are a group of polyphenols found in tea (Camellia sinensis var. sinensis) at high levels. They are beneficial for health. From the study on accumulation of catechins in shoots and mature leaves of a tea cultivar, Oolong No. 17, using high-performance liquid chromatography (HPLC), it was found that the amounts of most catechins in the shoots were higher than those in the mature leaves, with an exception of catechins gallate (CG) that was found in trace amounts in both the shoots and mature leaves. mRNA accumulation of genes involved in catechin synthesis was studied using reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that the mRNA accumulation of the genes were higher in the shoots than in the mature leaves. These genes included genes of phenylalanine ammonia-lyase 1 (PAL1; EC 4.3.1.5), chalcone synthase (CHS; EC 2.3.1.74), dihydroflavonol 4-reductase (DFR; EC 1.1.1.219), leucoanthocyanidin reductase (LCR; EC 1.17.1.3), and flavanone 3-hydroxylase (F3H; EC 1.14.11.9).

Rift Valley fever virus NS{sub S} gene expression correlates with a defect in nuclear mRNA export

DOE Office of Scientific and Technical Information (OSTI.GOV)

Copeland, Anna Maria; Van Deusen, Nicole M.; Schmaljohn, Connie S., E-mail: Connie.s.schmaljohn.civ@mail.mil

We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NS{sub S} gene, but not the N, G{sub N} or NS{sub M} genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NS{sub S}, confirming that expression of NS{sub S} is likely responsible for this phenomenon. - Highlights: • Riftmore » Valley fever virus (RVFV) infection alters the localization of host mRNA. • mRNA accumulates in the nuclei of RVFV-infected but not mock-infected cells. • NS{sub S} is likely responsible for mRNA relocalization to the nucleus.« less

Selection of the in vitro culture media influences mRNA expression of Hedgehog genes, Il-6, and important genes regarding reactive oxygen species in single murine preimplantation embryos.

PubMed

Pfeifer, N; Baston-Büst, D M; Hirchenhain, J; Friebe-Hoffmann, U; Rein, D T; Krüssel, J S; Hess, A P

2012-01-01

The aim of this paper was to determine the influence of different in vitro culture media on mRNA expression of Hedgehog genes, il-6, and important genes regarding reactive oxygen species in single mouse embryos. Reverse transcription of single embryos either cultured in vitro from day 0.5 until 3.5 (COOK's Cleavage medium or Vitrolife's G-1 PLUS medium) or in vivo until day 3.5 post coitum. PCR was carried out for β-actin followed by nested-PCR for shh, ihh, il-6, nox, gpx4, gpx1, and prdx2. The number of murine blastocysts cultured in COOK medium which expressed il-6, gpx4, gpx1, and prdx2 mRNA differed significantly compared to the in vivo group. Except for nox, the mRNA profile of the Vitrolife media group embryos varied significantly from the in vivo ones regarding the number of blastocysts expressing the mRNA of shh, ihh, il-6, gpx4, gpx1 and prdx2. The present study shows that different in vitro culture media lead to different mRNA expression profiles during early development. Even the newly developed in vitro culture media are not able to mimic the female reproductive tract. The question of long-term consequences for children due to assisted reproduction techniques needs to be addressed in larger studies.

Selection of the In Vitro Culture Media Influences mRNA Expression of Hedgehog Genes, Il-6, and Important Genes regarding Reactive Oxygen Species in Single Murine Preimplantation Embryos

PubMed Central

Pfeifer, N.; Baston-Büst, D. M.; Hirchenhain, J.; Friebe-Hoffmann, U.; Rein, D. T.; Krüssel, J. S.; Hess, A. P.

2012-01-01

Background. The aim of this paper was to determine the influence of different in vitro culture media on mRNA expression of Hedgehog genes, il-6, and important genes regarding reactive oxygen species in single mouse embryos. Methods. Reverse transcription of single embryos either cultured in vitro from day 0.5 until 3.5 (COOK's Cleavage medium or Vitrolife's G-1 PLUS medium) or in vivo until day 3.5 post coitum. PCR was carried out for β-actin followed by nested-PCR for shh, ihh, il-6, nox, gpx4, gpx1, and prdx2. Results. The number of murine blastocysts cultured in COOK medium which expressed il-6, gpx4, gpx1, and prdx2 mRNA differed significantly compared to the in vivo group. Except for nox, the mRNA profile of the Vitrolife media group embryos varied significantly from the in vivo ones regarding the number of blastocysts expressing the mRNA of shh, ihh, il-6, gpx4, gpx1 and prdx2. Conclusions. The present study shows that different in vitro culture media lead to different mRNA expression profiles during early development. Even the newly developed in vitro culture media are not able to mimic the female reproductive tract. The question of long-term consequences for children due to assisted reproduction techniques needs to be addressed in larger studies. PMID:22919324

Actin isoform and alpha 1B-adrenoceptor gene expression in aortic and coronary smooth muscle is influenced by cyclical stretch.

PubMed

Lundberg, M S; Sadhu, D N; Grumman, V E; Chilian, W M; Ramos, K S

1995-09-01

The occurrence of vascular domains with specific biological and pharmacological characteristics suggests that smooth muscle cells in different arteries may respond differentially to a wide range of environmental stimuli. To determine if some of these vessel-specific differences may be attributable to mechano-sensitive gene regulation, the influence of cyclical stretch on the expression of actin isoform and alpha 1B-adrenoceptor genes was examined in aortic and coronary smooth muscle cells. Cells were seeded on an elastin substrate and subjected to maximal stretching (24% elongation) and relaxation cycles at a frequency of 120 cycles/min in a Flexercell strain unit for 72 h. Total RNA was extracted and hybridized to radiolabeled cDNA probes to assess gene expression. Stretch caused a greater reduction of actin isoform mRNA levels in aortic smooth muscle cells as compared to cells from the coronary artery. Steady-state mRNA levels of alpha 1B-adrenoceptor were also decreased by cyclical stretch in both cell types but the magnitude of the response was greater in coronary smooth muscle cells. No changes in alpha 1B-adrenoceptor or beta/gamma-actin steady-state mRNA levels were observed in H4IIE cells, a nonvascular, immortalized cell line. The relative gene expression of heat shock protein 70 was not influenced by the cyclic stretch regimen in any of these cell types. These results suggest that stretch may participate in the regulation of gene expression in vascular smooth muscle cells and that this response exhibits some degree of cell-specificity.

High Efficiency CRISPR/Cas9-mediated Gene Editing in Primary Human T-cells Using Mutant Adenoviral E4orf6/E1b55k "Helper" Proteins.

PubMed

Gwiazda, Kamila S; Grier, Alexandra E; Sahni, Jaya; Burleigh, Stephen M; Martin, Unja; Yang, Julia G; Popp, Nicholas A; Krutein, Michelle C; Khan, Iram F; Jacoby, Kyle; Jensen, Michael C; Rawlings, David J; Scharenberg, Andrew M

2016-09-29

Many future therapeutic applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 and related RNA-guided nucleases are likely to require their use to promote gene targeting, thus necessitating development of methods that provide for delivery of three components-Cas9, guide RNAs and recombination templates-to primary cells rendered proficient for homology-directed repair. Here, we demonstrate an electroporation/transduction codelivery method that utilizes mRNA to express both Cas9 and mutant adenoviral E4orf6 and E1b55k helper proteins in association with adeno-associated virus (AAV) vectors expressing guide RNAs and recombination templates. By transiently enhancing target cell permissiveness to AAV transduction and gene editing efficiency, this novel approach promotes efficient gene disruption and/or gene targeting at multiple loci in primary human T-cells, illustrating its broad potential for application in translational gene editing.

Tryptophan derivatives regulate the transcription of Oct4 in stem-like cancer cells.

PubMed

Cheng, Jie; Li, Wenxin; Kang, Bo; Zhou, Yanwen; Song, Jiasheng; Dan, Songsong; Yang, Ying; Zhang, Xiaoqian; Li, Jingchao; Yin, Shengyong; Cao, Hongcui; Yao, Hangping; Zhu, Chenggang; Yi, Wen; Zhao, Qingwei; Xu, Xiaowei; Zheng, Min; Zheng, Shusen; Li, Lanjuan; Shen, Binghui; Wang, Ying-Jie

2015-06-10

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that responds to environmental toxicants, is increasingly recognized as a key player in embryogenesis and tumorigenesis. Here we show that a variety of tryptophan derivatives that act as endogenous AhR ligands can affect the transcription level of the master pluripotency factor Oct4. Among them, ITE enhances the binding of the AhR to the promoter of Oct4 and suppresses its transcription. Reduction of endogenous ITE levels in cancer cells by tryptophan deprivation or hypoxia leads to Oct4 elevation, which can be reverted by administration with synthetic ITE. Consequently, synthetic ITE induces the differentiation of stem-like cancer cells and reduces their tumorigenic potential in both subcutaneous and orthotopic xenograft tumour models. Thus, our results reveal a role of tryptophan derivatives and the AhR signalling pathway in regulating cancer cell stemness and open a new therapeutic avenue to target stem-like cancer cells.

Tryptophan derivatives regulate the transcription of Oct4 in stem-like cancer cells

PubMed Central

Cheng, Jie; Li, Wenxin; Kang, Bo; Zhou, Yanwen; Song, Jiasheng; Dan, Songsong; Yang, Ying; Zhang, Xiaoqian; Li, Jingchao; Yin, Shengyong; Cao, Hongcui; Yao, Hangping; Zhu, Chenggang; Yi, Wen; Zhao, Qingwei; Xu, Xiaowei; Zheng, Min; Zheng, Shusen; Li, Lanjuan; Shen, Binghui; Wang, Ying-Jie

2015-01-01

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that responds to environmental toxicants, is increasingly recognized as a key player in embryogenesis and tumorigenesis. Here we show that a variety of tryptophan derivatives that act as endogenous AhR ligands can affect the transcription level of the master pluripotency factor Oct4. Among them, ITE enhances the binding of the AhR to the promoter of Oct4 and suppresses its transcription. Reduction of endogenous ITE levels in cancer cells by tryptophan deprivation or hypoxia leads to Oct4 elevation, which can be reverted by administration with synthetic ITE. Consequently, synthetic ITE induces the differentiation of stem-like cancer cells and reduces their tumorigenic potential in both subcutaneous and orthotopic xenograft tumour models. Thus, our results reveal a role of tryptophan derivatives and the AhR signalling pathway in regulating cancer cell stemness and open a new therapeutic avenue to target stem-like cancer cells. PMID:26059097

Chitosan nanoparticle carrying small interfering RNA to platelet-derived growth factor B mRNA inhibits proliferation of smooth muscle cells in rabbit injured arteries.

PubMed

Xia, He; Jun, Ji; Wen-Ping, Ling; Yi-Feng, Pan; Xiao-Ling, Chen

2013-10-01

The purpose of this study was to elucidate the transfection of chitosan nanoparticle carrying small interfering RNA against platelet-derived growth factor B (PDGF-B) to inhibit the expression of PDGF-B mRNA and proliferation of smooth muscle cells. A rabbit iliac artery injury model was constructed. A small interfering RNA (siRNA) against PDGF-B mRNA expression vector was constructed and packaged by chitosan nanoparticle to transfect into the vascular smooth muscle cells (vSMCs) of balloon catheter-injured rabbit iliac artery wall, using a therapeutic ultrasound for the gene delivery. The experiment was divided into two groups: experimental group, denudation and nano-PDGF-B siRNA treated, and only single denudation as control. Effects of the siRNA on the expressions of proliferating cell nuclear antigen (PCNA) and PDGF-B mRNA by vSMCs and the proliferation of vSMCs were observed with the methods of routine pathological, immunohistochemical staining, in situ hybridization and morphometry. The nano siRNA against PDGF-B was successfully transfected. The nano siRNA significantly inhibited the expressions of PCNA and PDGF-B mRNA in intimal vSMCs. The local intimal thickness and area were also reduced remarkably. In conclusion, transfection of chitosan nanoparticle carrying siRNA against PDGF-B mRNA could inhibit proliferation of vSMCs in the rabbit iliac artery injury model. © The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

mRNA trans-splicing in gene therapy for genetic diseases.

PubMed

Berger, Adeline; Maire, Séverine; Gaillard, Marie-Claude; Sahel, José-Alain; Hantraye, Philippe; Bemelmans, Alexis-Pierre

2016-07-01

Spliceosome-mediated RNA trans-splicing, or SMaRT, is a promising strategy to design innovative gene therapy solutions for currently intractable genetic diseases. SMaRT relies on the correction of mutations at the post-transcriptional level by modifying the mRNA sequence. To achieve this, an exogenous RNA is introduced into the target cell, usually by means of gene transfer, to induce a splice event in trans between the exogenous RNA and the target endogenous pre-mRNA. This produces a chimeric mRNA composed partly of exons of the latter, and partly of exons of the former, encoding a sequence free of mutations. The principal challenge of SMaRT technology is to achieve a reaction as complete as possible, i.e., resulting in 100% repairing of the endogenous mRNA target. The proof of concept of SMaRT feasibility has already been established in several models of genetic diseases caused by recessive mutations. In such cases, in fact, the repair of only a portion of the mutant mRNA pool may be sufficient to obtain a significant therapeutic effect. However in the case of dominant mutations, the target cell must be freed from the majority of mutant mRNA copies, requiring a highly efficient trans-splicing reaction. This likely explains why only a few examples of SMaRT approaches targeting dominant mutations are reported in the literature. In this review, we explain in details the mechanism of trans-splicing, review the different strategies that are under evaluation to lead to efficient trans-splicing, and discuss the advantages and limitations of SMaRT. WIREs RNA 2016, 7:487-498. doi: 10.1002/wrna.1347 For further resources related to this article, please visit the WIREs website. © 2016 The Authors. WIREs RNA published by Wiley Periodicals, Inc.

SH3BP4, a novel pigmentation gene, is inversely regulated by miR-125b and MITF

PubMed Central

Kim, Kyu-Han; Lee, Tae Ryong; Cho, Eun-Gyung

2017-01-01

Our previous work has identified miR-125b as a negative regulator of melanogenesis. However, the specific melanogenesis-related genes targeted by this miRNA had not been identified. In this study, we established a screening strategy involving three consecutive analytical approaches—analysis of target genes of miR-125b, expression correlation analysis between each target gene and representative pigmentary genes, and functional analysis of candidate genes related to melanogenesis—to discover melanogenesis-related genes targeted by miR-125b. Through these analyses, we identified SRC homology 3 domain-binding protein 4 (SH3BP4) as a novel pigmentation gene. In addition, by combining bioinformatics analysis and experimental validation, we demonstrated that SH3BP4 is a direct target of miR-125b. Finally, we found that SH3BP4 is transcriptionally regulated by microphthalmia-associated transcription factor as its direct target. These findings provide important insights into the roles of miRNAs and their targets in melanogenesis. PMID:28819321

Aberrant EPHB4 gene methylation and childhood acute lymphoblastic leukemia

PubMed Central

Li, Yuhua; Wang, Huihui; Chen, Xiaowen; Mai, Huirong; Li, Changgang; Wen, Feiqiu

2017-01-01

The present study aimed to investigate the association between aberrant DNA methylation of the promoter region of the ephrin type-B receptor 4 (EPHB4) gene and the development of childhood acute lymphoblastic leukemia (ALL). Bisulfite sequencing polymerase chain reaction (BSP) was performed to determine the methylation density of cytosine-guanine pair islands in the promoter region of EPHB4, in bone marrow samples from 40 children with ALL. The mRNA and protein expression levels of EPHB4 were detected using reverse transcription-quantitative polymerase chain reaction and western blot analysis. A total of 10 children with idiopathic thrombocytopenic purpura (ITP) were recruited as controls. The results revealed that the average methylation density of the bone marrow samples from the patients with ALL was significantly higher, compared with the patients with ITP (P=0.046). The relative mRNA expression levels of EPHB4 in the patients with ITP (25.08±4.03) and the patients with ALL without methylation (12.33±2.16) were significantly higher, compared with that observed in the patients with ALL with methylation (6.48±2.73; P<0.01). Pearson analysis revealed a significant negative linear correlation between EPHB4 gene methylation and its expression levels (r=−0.957; P<0.01). Western blot analysis indicated that EPHB4 protein expression levels were low in the methylated ALL samples. An evaluation of the two-year disease-free survival (DFS) of the patients with ALL was performed, which revealed that the patients with unmethylated ALL exhibited a significantly higher two-year DFS rate, as compared with patients with methylated ALL (P=0.036). These results suggest that the methylation of the EPHB4 gene is prevalent in childhood ALL and may result in expressional inactivation, which consequently promotes ALL pathogenesis and is associated with an unfavorable prognosis. Therefore, the EPHB4 gene may function as a potential tumor suppressor in childhood ALL. PMID:29085439

Gold nanoparticle-based beacon to detect STAT5b mRNA expression in living cells: a case optimized by bioinformatics screen.

PubMed

Deng, Dawei; Li, Yang; Xue, Jianpeng; Wang, Jie; Ai, Guanhua; Li, Xin; Gu, Yueqing

2015-01-01

Messenger RNA (mRNA), a single-strand ribonucleic acid with functional gene information is usually abnormally expressed in cancer cells and has become a promising biomarker for the study of tumor progress. Hairpin DNA-coated gold nanoparticle (hDAuNP) beacon containing a bare gold nanoparticle (AuNP) as fluorescence quencher and thiol-terminated fluorescently labeled stem-loop-stem oligonucleotide sequences attached by Au-S bond is currently a new nanoscale biodiagnostic platform capable of mRNA detection, in which the design of the loop region sequence is crucial for hybridizing with the target mRNA. Hence, in this study, to improve the sensitivity and selectivity of hDAuNP beacon simultaneously, the loop region of hairpin DNA was screened by bioinformatics strategy. Here, signal transducer and activator of transcription 5b (STAT5b) mRNA was selected and used as a practical example. The results from the combined characterizations using optical techniques, flow cytometry assay, and cell microscopic imaging showed that after optimization, the as-prepared hDAuNP beacon had higher selectivity and sensitivity for the detection of STAT5b mRNA in living cells, as compared with our previous beacon. Thus, the bioinformatics method may be a promising new strategy for assisting in the designing of the hDAuNP beacon, extending its application in the detection of mRNA expression and the resultant mRNA-based biological processes and disease pathogenesis.

Gold nanoparticle-based beacon to detect STAT5b mRNA expression in living cells: a case optimized by bioinformatics screen

PubMed Central

Deng, Dawei; Li, Yang; Xue, Jianpeng; Wang, Jie; Ai, Guanhua; Li, Xin; Gu, Yueqing

2015-01-01

Messenger RNA (mRNA), a single-strand ribonucleic acid with functional gene information is usually abnormally expressed in cancer cells and has become a promising biomarker for the study of tumor progress. Hairpin DNA-coated gold nanoparticle (hDAuNP) beacon containing a bare gold nanoparticle (AuNP) as fluorescence quencher and thiol-terminated fluorescently labeled stem–loop–stem oligonucleotide sequences attached by Au–S bond is currently a new nanoscale biodiagnostic platform capable of mRNA detection, in which the design of the loop region sequence is crucial for hybridizing with the target mRNA. Hence, in this study, to improve the sensitivity and selectivity of hDAuNP beacon simultaneously, the loop region of hairpin DNA was screened by bioinformatics strategy. Here, signal transducer and activator of transcription 5b (STAT5b) mRNA was selected and used as a practical example. The results from the combined characterizations using optical techniques, flow cytometry assay, and cell microscopic imaging showed that after optimization, the as-prepared hDAuNP beacon had higher selectivity and sensitivity for the detection of STAT5b mRNA in living cells, as compared with our previous beacon. Thus, the bioinformatics method may be a promising new strategy for assisting in the designing of the hDAuNP beacon, extending its application in the detection of mRNA expression and the resultant mRNA-based biological processes and disease pathogenesis. PMID:25987838

Unmasking Upstream Gene Expression Regulators with miRNA-corrected mRNA Data

PubMed Central

Bollmann, Stephanie; Bu, Dengpan; Wang, Jiaqi; Bionaz, Massimo

2015-01-01

Expressed micro-RNA (miRNA) affects messenger RNA (mRNA) abundance, hindering the accuracy of upstream regulator analysis. Our objective was to provide an algorithm to correct such bias. Large mRNA and miRNA analyses were performed on RNA extracted from bovine liver and mammary tissue. Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%). Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%) and four levels of the magnitude of miRNA effect (ME) on mRNA expression (30%, 50%, 75%, and 83% mRNA reduction), we generated 17 different datasets (including the original dataset). For each dataset, we performed upstream regulator analysis using two bioinformatics tools. We detected an increased effect on the upstream regulator analysis with larger miRNA:mRNA pair bins and higher ME. The miRNA correction allowed identification of several upstream regulators not present in the analysis of the original dataset. Thus, the proposed algorithm improved the prediction of upstream regulators. PMID:27279737

Distinct types of primary cutaneous large B-cell lymphoma identified by gene expression profiling.

PubMed

Hoefnagel, Juliette J; Dijkman, Remco; Basso, Katia; Jansen, Patty M; Hallermann, Christian; Willemze, Rein; Tensen, Cornelis P; Vermeer, Maarten H

2005-05-01

In the European Organization for Research and Treatment of Cancer (EORTC) classification 2 types of primary cutaneous large B-cell lymphoma (PCLBCL) are distinguished: primary cutaneous follicle center cell lymphomas (PCFCCL) and PCLBCL of the leg (PCLBCL-leg). Distinction between both groups is considered important because of differences in prognosis (5-year survival > 95% and 52%, respectively) and the first choice of treatment (radiotherapy or systemic chemotherapy, respectively), but is not generally accepted. To establish a molecular basis for this subdivision in the EORTC classification, we investigated the gene expression profiles of 21 PCLBCLs by oligonucleotide microarray analysis. Hierarchical clustering based on a B-cell signature (7450 genes) classified PCLBCL into 2 distinct subgroups consisting of, respectively, 8 PCFCCLs and 13 PCLBCLsleg. PCLBCLs-leg showed increased expression of genes associated with cell proliferation; the proto-oncogenes Pim-1, Pim-2, and c-Myc; and the transcription factors Mum1/IRF4 and Oct-2. In the group of PCFCCL high expression of SPINK2 was observed. Further analysis suggested that PCFCCLs and PCLBCLs-leg have expression profiles similar to that of germinal center B-cell-like and activated B-cell-like diffuse large B-cell lymphoma, respectively. The results of this study suggest that different pathogenetic mechanisms are involved in the development of PCFCCLs and PCLBCLs-leg and provide molecular support for the subdivision used in the EORTC classification.

Quantifying Temporal Autocorrelations for the Expression of Geobacter species mRNA Gene Transcripts at Variable Ammonium Levels during in situ U(VI) Bioremediation

NASA Astrophysics Data System (ADS)

Mouser, P. J.

2010-12-01

In order to develop decision-making tools for the prediction and optimization of subsurface bioremediation strategies, we must be able to link the molecular-scale activity of microorganisms involved in remediation processes with biogeochemical processes observed at the field-scale. This requires the ability to quantify changes in the in situ metabolic condition of dominant microbes and associate these changes to fluctuations in nutrient levels throughout the bioremediation process. It also necessitates a need to understand the spatiotemporal variability of the molecular-scale information to develop meaningful parameters and constraint ranges in complex bio-physio-chemical models. The expression of three Geobacter species genes (ammonium transporter (amtB), nitrogen fixation (nifD), and a housekeeping gene (recA)) were tracked at two monitoring locations that differed significantly in ammonium (NH4+) concentrations during a field-scale experiment where acetate was injected into the subsurface to simulate Geobacteraceae in a uranium-contaminated aquifer. Analysis of amtB and nifD mRNA transcript levels indicated that NH4+ was the primary form of fixed nitrogen during bioremediation. Overall expression levels of amtB were on average 8-fold higher at NH4+ concentrations of 300 μM or more than at lower NH4+ levels (average 60 μM). The degree of temporal correlation in Geobacter species mRNA expression levels was calculated at both locations using autocorrelation methods that describe the relationship between sample semi-variance and time lag. At the monitoring location with lower NH4+, a temporal correlation lag of 8 days was observed for both amtB and nifD transcript patterns. At the location where higher NH4+ levels were observed, no discernable temporal correlation lag above the sampling frequency (approximately every 2 days) was observed for amtB or nifD transcript fluctuations. Autocorrelation trends in recA expression levels at both locations indicated that

OCT methods for capillary velocimetry

PubMed Central

Srinivasan, Vivek J.; Radhakrishnan, Harsha; Lo, Eng H.; Mandeville, Emiri T.; Jiang, James Y.; Barry, Scott; Cable, Alex E.

2012-01-01

To date, two main categories of OCT techniques have been described for imaging hemodynamics: Doppler OCT and OCT angiography. Doppler OCT can measure axial velocity profiles and flow in arteries and veins, while OCT angiography can determine vascular morphology, tone, and presence or absence of red blood cell (RBC) perfusion. However, neither method can quantify RBC velocity in capillaries, where RBC flow is typically transverse to the probe beam and single-file. Here, we describe new methods that potentially address these limitations. Firstly, we describe a complex-valued OCT signal in terms of a static scattering component, dynamic scattering component, and noise. Secondly, we propose that the time scale of random fluctuations in the dynamic scattering component are related to red blood cell velocity. Analysis was performed along the slow axis of repeated B-scans to parallelize measurements. We correlate our purported velocity measurements against two-photon microscopy measurements of RBC velocity, and investigate changes during hypercapnia. Finally, we image the ischemic stroke penumbra during distal middle cerebral artery occlusion (dMCAO), where OCT velocimetry methods provide additional insight that is not afforded by either Doppler OCT or OCT angiography. PMID:22435106

Overexpression of p53 mRNA in colorectal cancer and its relationship to p53 gene mutation.

PubMed Central

el-Mahdani, N.; Vaillant, J. C.; Guiguet, M.; Prévot, S.; Bertrand, V.; Bernard, C.; Parc, R.; Béréziat, G.; Hermelin, B.

1997-01-01

We analysed the frequency of p53 mRNA overexpression in a series of 109 primary colorectal carcinomas and its association with p53 gene mutation, which has been correlated with short survival. Sixty-nine of the 109 cases (63%) demonstrated p53 mRNA overexpression, without any correlation with stage or site of disease. Comparison with p53 gene mutation indicated that, besides cases in which p53 gene mutation and p53 mRNA overexpression were either both present (40 cases) or both absent (36 cases), there were also cases in which p53 mRNA was overexpressed in the absence of any mutation (29 cases) and those with a mutant gene in which the mRNA was not overexpressed (four cases). Moreover, the mutant p53 tumours exhibited an increase of p53 mRNA expression, which was significantly higher in tumours expressing the mutated allele alone than in tumours expressing both wild- and mutated-type alleles. These data (1) show that p53 mRNA overexpression is a frequent event in colorectal tumours and is not predictive of the status of the gene, i.e. whether or not a mutation is present; (2) provide further evidence that p53 protein overexpression does not only result from an increase in the half-life of mutated p53 and suggest that inactivation of the p53 function in colorectal cancers involves at least two distinct mechanisms, including p53 overexpression and/or mutation; and (3) suggest that p53 mRNA overexpression is an early event, since it is not correlated with Dukes stage. PMID:9052405

Cell cycle-dependent regulation of Aurora kinase B mRNA by the Microprocessor complex.

PubMed

Jung, Eunsun; Seong, Youngmo; Seo, Jae Hong; Kwon, Young-Soo; Song, Hoseok

2014-03-28

Aurora kinase B regulates the segregation of chromosomes and the spindle checkpoint during mitosis. In this study, we showed that the Microprocessor complex, which is responsible for the processing of the primary transcripts during the generation of microRNAs, destabilizes the mRNA of Aurora kinase B in human cells. The Microprocessor-mediated cleavage kept Aurora kinase B at a low level and prevented premature entrance into mitosis. The cleavage was reduced during mitosis leading to the accumulation of Aurora kinase B mRNA and protein. In addition to Aurora kinase B mRNA, the processing of other primary transcripts of miRNAs were also decreased during mitosis. We found that the cleavage was dependent on an RNA helicase, DDX5, and the association of DDX5 and DDX17 with the Microprocessor was reduced during mitosis. Thus, we propose a novel mechanism by which the Microprocessor complex regulates stability of Aurora kinase B mRNA and cell cycle progression. Copyright © 2014 Elsevier Inc. All rights reserved.

Identification and Characterization of Functionally Critical, Conserved Motifs in the Internal Repeats and N-terminal Domain of Yeast Translation Initiation Factor 4B (yeIF4B)*

PubMed Central

Zhou, Fujun; Walker, Sarah E.; Mitchell, Sarah F.; Lorsch, Jon R.; Hinnebusch, Alan G.

2014-01-01

eIF4B has been implicated in attachment of the 43 S preinitiation complex (PIC) to mRNAs and scanning to the start codon. We recently determined that the internal seven repeats (of ∼26 amino acids each) of Saccharomyces cerevisiae eIF4B (yeIF4B) compose the region most critically required to enhance mRNA recruitment by 43 S PICs in vitro and stimulate general translation initiation in yeast. Moreover, although the N-terminal domain (NTD) of yeIF4B contributes to these activities, the RNA recognition motif is dispensable. We have now determined that only two of the seven internal repeats are sufficient for wild-type (WT) yeIF4B function in vivo when all other domains are intact. However, three or more repeats are needed in the absence of the NTD or when the functions of eIF4F components are compromised. We corroborated these observations in the reconstituted system by demonstrating that yeIF4B variants with only one or two repeats display substantial activity in promoting mRNA recruitment by the PIC, whereas additional repeats are required at lower levels of eIF4A or when the NTD is missing. These findings indicate functional overlap among the 7-repeats and NTD domains of yeIF4B and eIF4A in mRNA recruitment. Interestingly, only three highly conserved positions in the 26-amino acid repeat are essential for function in vitro and in vivo. Finally, we identified conserved motifs in the NTD and demonstrate functional overlap of two such motifs. These results provide a comprehensive description of the critical sequence elements in yeIF4B that support eIF4F function in mRNA recruitment by the PIC. PMID:24285537

Interleukin-1 homologues IL-1F7b and IL-18 contain functional mRNA instability elements within the coding region responsive to lipopolysaccharide

PubMed Central

2004-01-01

IL-1F7b, a novel homologue of the IL-1 (interleukin 1) family, was discovered by computational cloning. We demonstrated that IL-1F7b shares critical amino acid residues with IL-18 and binds to the IL-18-binding protein enhancing its ability to inhibit IL-18-induced interferon-γ. We also showed that low levels of IL-1F7b are constitutively present intracellularly in human blood monocytes. In this study, we demonstrate that similar to IL-18, both mRNA and intracellular protein expression of IL-1F7b are up-regulated by LPS (lipopolysaccharide) in human monocytes. In stable transfectants of murine RAW264.7 macrophage cells, there was no IL-1F7b protein expression despite a highly active CMV promoter. We found that IL-1F7b-specific mRNA was rapidly degraded in transfected cells, via a 3′-UTR (untranslated region)-independent control of IL-1F7b transcript stability. After LPS stimulation, there was a rapid transient increase in IL-1F7b-specific mRNA and concomitant protein levels. Using sequence alignment, we found a conserved ten-nucleotide homology box within the open reading frame of IL-F7b, which is flanking the coding region instability elements of some selective genes. In-frame deletion of downstream exon 5 from the full-length IL-1F7b cDNA markedly increased the levels of IL-1F7b mRNA. A similar coding region element is located in IL-18. When transfected into RAW264.7 macrophages, IL-18 mRNA was also unstable unless treated with LPS. These results indicate that both IL-1F7b and IL-18 mRNA contain functional instability determinants within their coding region, which influence mRNA decay as a novel mechanism to regulate the expression of IL-1 family members. PMID:15046617

eIF4B stimulates translation of long mRNAs with structured 5′ UTRs and low closed-loop potential but weak dependence on eIF4G

PubMed Central

Sen, Neelam Dabas; Zhou, Fujun; Harris, Michael S.; Ingolia, Nicholas T.

2016-01-01

DEAD-box RNA helicases eukaryotic translation initiation factor 4A (eIF4A) and Ded1 promote translation by resolving mRNA secondary structures that impede preinitiation complex (PIC) attachment to mRNA or scanning. Eukaryotic translation initiation factor 4B (eIF4B) is a cofactor for eIF4A but also might function independently of eIF4A. Ribosome profiling of mutants lacking eIF4B or with impaired eIF4A or Ded1 activity revealed that eliminating eIF4B reduces the relative translational efficiencies of many more genes than does inactivation of eIF4A, despite comparable reductions in bulk translation, and few genes display unusually strong requirements for both factors. However, either eliminating eIF4B or inactivating eIF4A preferentially impacts mRNAs with longer, more structured 5′ untranslated regions (UTRs). These findings reveal an eIF4A-independent role for eIF4B in addition to its function as eIF4A cofactor in promoting PIC attachment or scanning on structured mRNAs. eIF4B, eIF4A, and Ded1 mutations also preferentially impair translation of longer mRNAs in a fashion mitigated by the ability to form closed-loop messenger ribonucleoprotein particles (mRNPs) via eIF4F–poly(A)-binding protein 1 (Pab1) association, suggesting cooperation between closed-loop assembly and eIF4B/helicase functions. Remarkably, depleting eukaryotic translation initiation factor 4G (eIF4G), the scaffold subunit of eukaryotic translation initiation factor 4F (eIF4F), preferentially impacts short mRNAs with strong closed-loop potential and unstructured 5′ UTRs, exactly the opposite features associated with hyperdependence on the eIF4B/helicases. We propose that short, highly efficient mRNAs preferentially depend on the stimulatory effects of eIF4G-dependent closed-loop assembly. PMID:27601676

The effect of extracellular alkalinization on lactate metabolism of breast cancer stem cells: Overview of LDH-A, LDH-B, MCT1 and MCT4 gene expression

NASA Astrophysics Data System (ADS)

Neolaka, G. M. G.; Yustisia, I.; Sadikin, M.; Wanandi, S. I.

2017-08-01

Changes in the metabolic status of cancer cells are presumed to be correlated with the adjustment of these cells to extracellular changes. Cell glycolysis increases the production of intracellular lactate catalyzed by the lactate dehydrogenases, both LDH-A and LDH-B. An increase in intracellular lactate can affect extracellular pH balance through monocarboxylate transporters, particularly MCT1 and MCT4. This study aimed to analyze the effects of extracellular alkalinization on the lactate metabolism of human breast cancer stem cells (BCSCs). In this study, human primary BCSCs (CD24-/CD44+ cells) were treated with 100 mM sodium bicarbonate for 0.5, 24, and 48 h in DMEM F12/HEPES. After incubation, extracellular pH was measured and cells were harvested to extract the total RNA and protein. The expression of LDH-A, LDH-B, MCT1, and MCT4 mRNA genes were analyzed using qRT-PCR method. Our study shows that administration of sodium bicarbonate in the BCSC culture medium could increase extracellular pH. To balance the increase of extracellular pH, BCSCs regulated the expression of LDH-A, LDH-B, MCT1, and MCT4 genes. As the extracellular pH increases, the expression of LDH-A that converts pyruvate to lactate increased along with the increase of MCT 4 and MCT 1 expression, which act as lactate transporters. As the incubation time increases, the pH decreases, leading to the suppression of LDH-A and increase of LDH-B expression that converts lactate into pyruvate. Therefore, we suggest that the extracellular alkalinization by sodium bicarbonate in BCSCs affected the genes that regulate lactate metabolism.

Multifunctional triblock co-polymer mP3/4HB-b-PEG-b-lPEI for efficient intracellular siRNA delivery and gene silencing.

PubMed

Zhou, Li; Chen, Zhifei; Wang, Feifei; Yang, Xiuqun; Zhang, Biliang

2013-04-01

A non-viral siRNA carrier composed of mono-methoxy-poly (3-hydroxybutyrate-co-4-hydroxybutyrate)-block-polyethylene glycol-block-linear polyethyleneimine (mP3/4HB-b-PEG-b-lPEI) was synthesized using 1800 Da linear polyethyleneimine and evaluated for siRNA delivery. Our study demonstrated that siRNA could be efficiently combined with mP3/4HB-b-PEG-b-lPEI (mAG) co-polymer and was protected from nuclease degradation. The combined siRNA were released from the complexes easily under heparin competition. The particle size of the mAG/siRNA complexes was 158 nm, with a ζ-potential of around 28 mV. Atomic force microscopy images displayed spherical and homogeneously distributed complexes. The mAG block co-polymer displayed low cytotoxicity and efficient cellular uptake of Cy3-siRNA in A549 cells by flow cytometry and confocal microscopy. In vitro transfection efficiency of the block co-polymer was assessed using siRNA against luciferase in cultured A549-Luc, HeLa-Luc, HLF-Luc, A375-Luc and MCF-7-Luc cells. A higher transfection efficiency and lower cytotoxicity was obtained by mAG block co-polymer in five cell lines. Furthermore, a remarkable improvement in luciferase gene silencing efficiency of the mAG complex (up to 90-95%) over that of Lipofectamine™ 2000 (70-82%) was observed in HLF-Luc and A375-Luc cells. Additionally, a mAG/p65-siRNA complex also showed a better capability than Lipofectamine™ 2000/p65-siRNA complex to drastically reduce the p65 mRNA level down to 10-16% in HeLa, U251 and HUVEC cells at an N/P ratio of 70. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

Zona pellucida gene mRNA expression in human oocytes is related to oocyte maturity, zona inner layer retardance and fertilization competence.

PubMed

Canosa, S; Adriaenssens, T; Coucke, W; Dalmasso, P; Revelli, A; Benedetto, C; Smitz, J

2017-05-01

Do the mRNA expression levels of zona pellucida (ZP) genes, ZP1, 2, 3 and 4 in oocyte and cumulus cells (CC) reveal relevant information on the oocyte? The ZP mRNA expression in human oocytes is related to oocyte maturity, zona inner layer (IL) retardance and fertilization capacity. ZP structure and birefringence provide useful information on oocyte cytoplasmic maturation, developmental competence for embryonic growth, blastocyst formation and pregnancy. In order to understand the molecular basis of morphological changes in the ZP, in the current study, the polarized light microscopy (PLM) approach was combined with analysis of the expression of the genes encoding ZP1, 2, 3 and 4, both in the oocytes and in the surrounding CC. This is a retrospective study comprising 98 supernumerary human cumulus oocyte complexes (COC) [80 Metaphase II (MII), 10 Metaphase I (MI) and 8 germinal vesicle (GV)] obtained from 39 patients (median age 33.4 years, range 22-42) after controlled ovarian stimulation. Single oocytes and their corresponding CC were analysed. Oocytes were examined using PLM, and quantitative RT-PCR was performed for ZP1, 2, 3 and 4 in these individual oocytes and their CC. Ephrin-B2 (EFNB2) mRNA was measured in CC as a control. Presence of ZP3 protein in CC and oocytes was investigated using immunocytochemistry. Data were analysed using one-parametric and multivariate analysis and were corrected for the potential impact of patient and cycle characteristics. Oocytes contained ZP1/2/3 and 4 mRNA while in CC only ZP3 was quantifiable. Also ZP3 protein was detected in human CC. When comparing mature (MII) and immature oocytes (MI/GV) or their corresponding CC, ZP1/2 and 4 expression was lower in mature oocytes compared to the expression in immature oocytes (all P < 0.05) and ZP3 expression was lower in the CC of mature oocytes compared to the expression in CC of immature oocytes (P < 0.05). This coincided with a significantly smaller IL-ZP area and thickness in

OCA-B regulation of B-cell development and function.

PubMed

Teitell, Michael A

2003-10-01

The transcriptional co-activator OCA-B [for Oct co-activator from B cells, also known as OBF-1 (OCT-binding factor-1) and Bob1] is not required for B-cell genesis but does regulate subsequent B-cell development and function. OCA-B deficient mice show strain-specific, partial blocks at multiple stages of B-cell maturation and a complete disruption of germinal center formation in all strains, causing humoral immune deficiency and susceptibility to infection. OCA-B probably exerts its effects through the regulation of octamer-motif controlled gene expression. The OCA-B gene encodes two proteins of distinct molecular weight, designated p34 and p35. The p34 isoform localizes in the nucleus, whereas the p35 isoform is myristoylated and is bound to the cytoplasmic membrane. p35 can traffic to the nucleus and probably activates octamer-dependent transcription, although this OCA-B isoform might regulate B cells through membrane-related signal transduction.

Structural and mechanistic insights into nuclear transport and delivery of the critical pluripotency factor Oct4 to DNA.

PubMed

Okuyama, Takahide; Yamagishi, Ryosuke; Shimada, Jiro; Ikeda, Masaaki; Maruoka, Yayoi; Kaneko, Hiroki

2018-02-01

Oct4 is a master regulator of the induction and maintenance of cellular pluripotency, and has crucial roles in early stages of differentiation. It is the only factor that cannot be substituted by other members of the same protein family to induce pluripotency. However, although Oct4 nuclear transport and delivery to target DNA are critical events for reprogramming to pluripotency, little is known about the molecular mechanism. Oct4 is imported to the nucleus by the classical nuclear transport mechanism, which requires importin α as an adaptor to bind the nuclear localization signal (NLS). Although there are structures of complexes of the NLS of transcription factors (TFs) in complex with importin α, there are no structures available for complexes involving intact TFs. We have therefore modeled the structure of the complex of the whole Oct4 POU domain and importin α2 using protein-protein docking and molecular dynamics. The model explains how the Ebola virus VP24 protein has a negative effect on the nuclear import of STAT1 by importin α but not on Oct4, and how Nup 50 facilitates cargo release from importin α. The model demonstrates the structural differences between the Oct4 importin α bound and DNA bound crystal states. We propose that the 'expanded linker' between the two DNA-binding domains of Oct4 is an intrinsically disordered region and that its conformational changes have a key role in the recognition/binding to both DNA and importin α. Moreover, we propose that this structural change enables efficient delivery to DNA after release from importin α.

The cellular growth rate controls overall mRNA turnover, and modulates either transcription or degradation rates of particular gene regulons.

PubMed

García-Martínez, José; Delgado-Ramos, Lidia; Ayala, Guillermo; Pelechano, Vicent; Medina, Daniel A; Carrasco, Fany; González, Ramón; Andrés-León, Eduardo; Steinmetz, Lars; Warringer, Jonas; Chávez, Sebastián; Pérez-Ortín, José E

2016-05-05

We analyzed 80 different genomic experiments, and found a positive correlation between both RNA polymerase II transcription and mRNA degradation with growth rates in yeast. Thus, in spite of the marked variation in mRNA turnover, the total mRNA concentration remained approximately constant. Some genes, however, regulated their mRNA concentration by uncoupling mRNA stability from the transcription rate. Ribosome-related genes modulated their transcription rates to increase mRNA levels under fast growth. In contrast, mitochondria-related and stress-induced genes lowered mRNA levels by reducing mRNA stability or the transcription rate, respectively. We also detected these regulations within the heterogeneity of a wild-type cell population growing in optimal conditions. The transcriptomic analysis of sorted microcolonies confirmed that the growth rate dictates alternative expression programs by modulating transcription and mRNA decay.The regulation of overall mRNA turnover keeps a constant ratio between mRNA decay and the dilution of [mRNA] caused by cellular growth. This regulation minimizes the indiscriminate transmission of mRNAs from mother to daughter cells, and favors the response capacity of the latter to physiological signals and environmental changes. We also conclude that, by uncoupling mRNA synthesis from decay, cells control the mRNA abundance of those gene regulons that characterize fast and slow growth. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The NO signaling pathway differentially regulates KCC3a and KCC3b mRNA expression.

PubMed

Di Fulvio, Mauricio; Lauf, Peter K; Adragna, Norma C

2003-11-01

Nitric oxide (NO) donors and protein kinase G (PKG) acutely up-regulate K-Cl cotransporter-1 and -3 (KCC1 and KCC3) mRNA expression in vascular smooth muscle cells (VSMCs). Here, we report the presence, relative abundance, and regulation by sodium nitroprusside (SNP) of the novel KCC3a and KCC3b mRNAs, in primary cultures of rat VSMCs. KCC3a and KCC3b mRNAs were expressed in an approximate 3:1 ratio, as determined by semiquantitative RT-PCR analysis. SNP as well as YC-1 and 8-Br-cGMP, a NO-independent stimulator of soluble guanylyl cyclase (sGC) and PKG, respectively, increased KCC3a and KCC3b mRNA expression by 2.5-fold and 8.1-fold in a time-dependent manner, following a differential kinetics. Stimulation of the NO/sGC/PKG signaling pathway with either SNP, YC-1, or 8-Br-cGMP decreased the KCC3a/KCC3b ratio from 3.0+/-0.4 to 0.9+/-0.1. This is the first report on a differential regulation by the NO/sGC/PKG signaling pathway of a cotransporter and of KCC3a and KCC3b mRNA expression.

Identification of a reference gene for the quantification of mRNA and miRNA expression during skin wound healing.

PubMed

Etich, Julia; Bergmeier, Vera; Pitzler, Lena; Brachvogel, Bent

2017-03-01

Wound healing is a coordinated process to restore tissue homeostasis and reestablish the protective barrier of the skin. miRNAs may modulate the expression of target genes to contribute to repair processes, but due to the complexity of the tissue it is challenging to quantify gene expression during the distinct phases of wound repair. Here, we aimed to identify a common reference gene to quantify changes in miRNA and mRNA expression during skin wound healing. Quantitative real-time PCR and bioinformatic analysis tools were used to identify suitable reference genes during skin repair and their reliability was tested by studying the expression of mRNAs and miRNAs. Morphological assessment of wounds showed that the injury model recapitulates the distinct phases of skin repair. Non-degraded RNA could be isolated from skin and wounds and used to study the expression of non-coding small nuclear RNAs during wound healing. Among those, RNU6B was most constantly expressed during skin repair. Using this reference gene we could confirm the transient upregulation of IL-1β and PTPRC/CD45 during the early phase as well as the increased expression of collagen type I at later stages of repair and validate the differential expression of miR-204, miR-205, and miR-31 in skin wounds. In contrast to Gapdh the normalization to multiple reference genes gave a similar outcome. RNU6B is an accurate alternative normalizer to quantify mRNA and miRNA expression during the distinct phases of skin wound healing when analysis of multiple reference genes is not feasible.

On-chip Mach-Zehnder interferometer for OCT systems

NASA Astrophysics Data System (ADS)

van Leeuwen, Ton G.; Akca, Imran B.; Angelou, Nikolaos; Weiss, Nicolas; Hoekman, Marcel; Leinse, Arne; Heideman, Rene G.

2018-04-01

By using integrated optics, it is possible to reduce the size and cost of a bulky optical coherence tomography (OCT) system. One of the OCT components that can be implemented on-chip is the interferometer. In this work, we present the design and characterization of a Mach-Zehnder interferometer consisting of the wavelength-independent splitters and an on-chip reference arm. The Si3N4 was chosen as the material platform as it can provide low losses while keeping the device size small. The device was characterized by using a home-built swept source OCT system. A sensitivity value of 83 dB, an axial resolution of 15.2 μm (in air) and a depth range of 2.5 mm (in air) were all obtained.

4D microscope-integrated OCT improves accuracy of ophthalmic surgical maneuvers

NASA Astrophysics Data System (ADS)

Carrasco-Zevallos, Oscar; Keller, Brenton; Viehland, Christian; Shen, Liangbo; Todorich, Bozho; Shieh, Christine; Kuo, Anthony; Toth, Cynthia; Izatt, Joseph A.

2016-03-01

Ophthalmic surgeons manipulate micron-scale tissues using stereopsis through an operating microscope and instrument shadowing for depth perception. While ophthalmic microsurgery has benefitted from rapid advances in instrumentation and techniques, the basic principles of the stereo operating microscope have not changed since the 1930's. Optical Coherence Tomography (OCT) has revolutionized ophthalmic imaging and is now the gold standard for preoperative and postoperative evaluation of most retinal and many corneal procedures. We and others have developed initial microscope-integrated OCT (MIOCT) systems for concurrent OCT and operating microscope imaging, but these are limited to 2D real-time imaging and require offline post-processing for 3D rendering and visualization. Our previously presented 4D MIOCT system can record and display the 3D surgical field stereoscopically through the microscope oculars using a dual-channel heads-up display (HUD) at up to 10 micron-scale volumes per second. In this work, we show that 4D MIOCT guidance improves the accuracy of depth-based microsurgical maneuvers (with statistical significance) in mock surgery trials in a wet lab environment. Additionally, 4D MIOCT was successfully performed in 38/45 (84%) posterior and 14/14 (100%) anterior eye human surgeries, and revealed previously unrecognized lesions that were invisible through the operating microscope. These lesions, such as residual and potentially damaging retinal deformation during pathologic membrane peeling, were visualized in real-time by the surgeon. Our integrated system provides an enhanced 4D surgical visualization platform that can improve current ophthalmic surgical practice and may help develop and refine future microsurgical techniques.

Expression of the tumor suppressor genes NF2, 4.1B, and TSLC1 in canine meningiomas.

PubMed

Dickinson, P J; Surace, E I; Cambell, M; Higgins, R J; Leutenegger, C M; Bollen, A W; LeCouteur, R A; Gutmann, D H

2009-09-01

Meningiomas are common primary brain tumors in dogs; however, little is known about the molecular genetic mechanisms involved in their tumorigenesis. Several tumor suppressor genes have been implicated in meningioma pathogenesis in humans, including the neurofibromatosis 2 (NF2), protein 4.1B (4.1 B), and tumor suppressor in lung cancer-1 (TSLC1) genes. We investigated the expression of these tumor suppressor genes in a series of spontaneous canine meningiomas using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) (NF2; n = 25) and western blotting (NF2/merlin, 4.1B, TSLC1; n = 30). Decreased expression of 4.1B and TSLC1 expression on western blotting was seen in 6/30 (20%) and in 15/30 (50%) tumors, respectively, with 18/30 (60%) of meningiomas having decreased or absent expression of one or both proteins. NF2 gene expression assessed by western blotting and RT-PCR varied considerably between individual tumors. Complete loss of NF2 protein on western blotting was not seen, unlike 4.1B and TSLC1. Incidence of TSLC1 abnormalities was similar to that seen in human meningiomas, while perturbation of NF2 and 4.1B appeared to be less common than reported for human tumors. No association was observed between tumor grade, subtype, or location and tumor suppressor gene expression based on western blot or RT-PCR. These results suggest that loss of these tumor suppressor genes is a frequent occurrence in canine meningiomas and may be an early event in tumorigenesis in some cases. In addition, it is likely that other, as yet unidentified, genes play an important role in canine meningioma formation and growth.

Successful TB treatment induces B-cells expressing FASL and IL5RA mRNA.

PubMed

van Rensburg, Ilana C; Wagman, Chandre; Stanley, Kim; Beltran, Caroline; Ronacher, Katharina; Walzl, Gerhard; Loxton, Andre G

2017-01-10

Activated B-cells increase T-cell behaviour during autoimmune disease and other infections by means of cytokine production and antigen-presentation. Functional studies in experimental autoimmune encephalomyelitis (EAE) indicate that B-cell deficiencies, and a lack of IL10 and IL35 leads to a poor prognosis. We hypothesised that B-cells play a role during tuberculosis. We evaluated B-cell mRNA expression using real-time PCR from healthy community controls, individuals with other lung diseases and newly diagnosed untreated pulmonary TB patients at three different time points (diagnosis, month 2 and 6 of treatment).We show that FASLG, IL5RA, CD38 and IL4 expression was lower in B-cells from TB cases compared to healthy controls. The changes in expression levels of CD38 may be due to a reduced activation of B-cells from TB cases at diagnosis. By month 2 of treatment, there was a significant increase in the expression of APRIL and IL5RA in TB cases. Furthermore, after 6 months of treatment, APRIL, FASLG, IL5RA and CD19 were upregulated in B-cells from TB cases. The increase in the expression of APRIL and CD19 suggests that there may be restored activation of B-cells following anti-TB treatment. The upregulation of FASLG and IL5RA indicates that B-cells expressing regulatory genes may play an important role in the protective immunity against M.tb infection. Our results show that increased activation of B-cells is present following successful TB treatment, and that the expression of FASLG and IL5RA could potentially be utilised as a signature to monitor treatment response.

Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trempe, J.P.; Carter, B.J.

1988-01-01

The authors studied the effects of the adeno-associated virus (AAV) rep gene on the control of gene expression from the AAV p/sub 40/ promoter in 293 cells in the absence of an adenovirus coinfection. AAV vectors containing the chloramphenicol acetyltransferase (cat) gene were used to measure the levels of cat expression and steady-state mRNA from p/sub 40/. When the rep gene was present in cis or in trans, cat expression from p/sub 40/ was decreased 3- to 10-fold, but there was a 2- to 10-fold increase in the level of p/sub 40/ mRNA. Conversely, cat expression increased and the p/submore » 40/ mRNA level decreased in the absence of the rep gene. Both wild-type and carboxyl-terminal truncated Rep proteins were capable of eliciting both effects. These data suggest two roles for the pleiotropic AAV rep gene: as a translational inhibitor and as a positive regulator of p/sub 40/ mRNA levels. They also provide additional evidence for a cis-acting negative regulatory region which decreases RNA from the AAV p/sub 5/ promoter in a fashion independent of rep.« less

Pertussis toxin export genes are regulated by the ptx promoter and may be required for efficient translation of ptx mRNA in Bordetella pertussis.

PubMed Central

Baker, S M; Masi, A; Liu, D F; Novitsky, B K; Deich, R A

1995-01-01

The gene products from an 8-kb region adjacent to the 3' end of the ptx operon are required by Bordetella pertussis for the export of pertussis holotoxin. At least one of these gene products (PtlC) is specifically required for the export of assembled holotoxin from the periplasmic space. ptlC mutants exhibit a 20-fold reduction in the amount of holotoxin present in the culture supernatant but have no effect upon the assembly or steady-state level of holotoxin present in the periplasmic space. Impaired export of holotoxin from the ptlC strain blocks expression of toxin at a posttranscriptional level, and wild-type levels of ptx mRNA are detected in the mutant strain. The transcription of ptl is subject to modulation by MgSO4 in the same manner as ptx is; however, in B. pertussis strains containing an E. coli tac promoter in place of the native ptx promoter, wild-type levels of ptx mRNA are present and holotoxin is synthesized and exported even in the presence of MgSO4. Promoter mapping of the region extending from the ptxS3 coding region to the ptlC coding region failed to detect the ptl transcription initiation site. Additional RNase protection experiments with ptx promoter deletion and substitution strains indicate that the ptl operon is transcribed from the ptx promoter as part of a > 11-kb mRNA. PMID:7558300

Heterogeneity in Oct4 and Sox2 Targets Biases Cell Fate in 4-Cell Mouse Embryos.

PubMed

Goolam, Mubeen; Scialdone, Antonio; Graham, Sarah J L; Macaulay, Iain C; Jedrusik, Agnieszka; Hupalowska, Anna; Voet, Thierry; Marioni, John C; Zernicka-Goetz, Magdalena

2016-03-24

The major and essential objective of pre-implantation development is to establish embryonic and extra-embryonic cell fates. To address when and how this fundamental process is initiated in mammals, we characterize transcriptomes of all individual cells throughout mouse pre-implantation development. This identifies targets of master pluripotency regulators Oct4 and Sox2 as being highly heterogeneously expressed between blastomeres of the 4-cell embryo, with Sox21 showing one of the most heterogeneous expression profiles. Live-cell tracking demonstrates that cells with decreased Sox21 yield more extra-embryonic than pluripotent progeny. Consistently, decreasing Sox21 results in premature upregulation of the differentiation regulator Cdx2, suggesting that Sox21 helps safeguard pluripotency. Furthermore, Sox21 is elevated following increased expression of the histone H3R26-methylase CARM1 and is lowered following CARM1 inhibition, indicating the importance of epigenetic regulation. Therefore, our results indicate that heterogeneous gene expression, as early as the 4-cell stage, initiates cell-fate decisions by modulating the balance of pluripotency and differentiation. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Vitamin D3 analog maxacalcitol (OCT) induces hCAP-18/LL-37 production in human oral epithelial cells.

PubMed

Tada, Hiroyuki; Shimizu, Takamitsu; Nagaoka, Isao; Takada, Haruhiko

2016-01-01

Maxacalcitol (22-oxacalcitriol: OCT) is a synthetic vitamin D3 analog with a limited calcemic effect. In this study, we investigated whether OCT increases the production of LL-37/CAP-18, a human cathelicidin antimicrobial peptide, in human gingival/oral epithelial cells. A human gingival epithelial cell line (Ca9-22) and human oral epithelial cell lines (HSC-2, HSC-3, and HSC-4) exhibited the enhanced expression of LL-37 mRNA upon stimulation with OCT as well as active metabolites of vitamins D3 and D2. Among the human epithelial cell lines, Ca9-22 exhibited the strongest response to these vitamin D-related compounds. OCT induced the higher production of CAP-18 (ng/mL order) until 6 days time-dependently in Ca9-22 cells in culture. The periodontal pathogen Porphyromonas gingivalis was killed by treatment with the LL-37 peptide. These findings suggest that OCT induces the production of hCAP-18/LL-37 in a manner similar to that induced by the active metabolite of vitamin D3.

Effects of postprandial starvation on mRNA expression of endocrine-, amino acid and peptide transporter-, and metabolic enzyme-related genes in zebrafish (Danio rerio).

PubMed

Tian, Juan; He, Gen; Mai, Kangsen; Liu, Chengdong

2015-06-01

The goal of this study was to systematically evaluate the molecular activities of endocrine-, amino acid and peptide transporters-, and metabolic enzyme-related genes in 35-day-old mixed-sex zebrafish (Danio rerio) after feeding . Zebrafish with initial body weights ranging from 9 to 11 mg were fasted for 384 h in a controlled indoor environment. Fish were sampled at 0, 3, 6, 12, 24, 48, 96, 192, and 384 h after fed. Overall, the present study results show that the regulatory mechanism that insulin-like growth factor I negative feedback regulated growth hormone is conserved in zebrafish, as it is in mammals, but that regulation of growth hormone receptors is highly intricate. Leptin and cholecystokinin are time-dependent negative feedback signals, and neuropeptide Y may be an important positive neuropeptide for food intake in zebrafish. The amino acid/carnitine transporters B(0,+) (ATB(0,+)) and broad neutral (0) amino acid transporter 1(B(0)AT1) mRNA levels measured in our study suggest that protein may be utilized during 24-96 h of fasting in zebrafish. Glutamine synthetase mRNA levels were downregulated, and glutamate dehydrogenase, alanine aminotransferase, aspartate transaminase, and trypsin mRNA levels were upregulated after longtime fasting in this study. The mRNA expression levels of fatty acid synthetase decreased significantly (P < 0.05), whereas those of lipoprotein lipase rapidly increased after 96 h of fasting. Fasting activated the expression of glucose synthesis genes when fasting for short periods of time; when fasting is prolonged, the mRNA levels of glucose breakdown enzymes and pentose phosphate shunt genes decreased.

Quantitation of CYP1A1 and 1B1 mRNA in polycyclic aromatic hydrocarbon-treated human T-47D and HepG2 cells by a modified bDNA assay using fluorescence detection.

PubMed

Wu, Susan J; Spink, David C; Spink, Barbara C; Kaminsky, Laurence S

2003-01-15

The quantitation of mRNA, essential for assessing mechanisms of enzyme regulation, is normally carried out using reverse transcriptase-polymerase chain reaction (RT-PCR). An alternative method uses a signal-amplification nucleic acid probe assay, which measures RNA directly by the QuantiGene Expression Kit and incorporates branched DNA technology from Bayer and luminometer-based readings of a chemilumigenic alkaline phosphatase substrate. To broaden the utility of this assay, we investigated substitution of a fluorescent substrate, 2'-(2-benzothiazol)-6'-hydroxybenzothiazole phosphate and a fluorometer, and applied the method to quantitation of CYP1A1 and 1B1 mRNA in human T-47D and HepG2 cells following induction by benzo[a]pyrene (B[a]P) and dibenzo[a,h]anthracene (DB[a,h]A). The fluorescence response increased linearly for 200 min without photobleaching and increased linearly (r2=0.997) up to at least 0.2 microg total RNA. The data revealed that at 0.5 and 1.0 microM inducing agent, the induction of CYP1A1 mRNA in HepG2 cells by DB[a,h]A exceeded that by B[a]P by 18- and 6-fold, respectively. In T-47D cells B[a]P induced CYP1A1 mRNA by 23-fold and CYP1B1 mRNA by 3.9-fold. A B[a]P cocontaminant in the environment, arsenite, did not affect B[a]P-induced levels of CYP1A1 or 1B1 mRNA in these cells. The modified analytical system provides a rapid-throughput, reproducible, and less labor-intensive method than RT-PCR for quantifying cellular mRNA levels.

In vitro culture of bovine embryos in murine ES cell conditioned media negatively affects expression of pluripotency-related markers OCT4, SOX2 and SSEA1.

PubMed

Oliveira, C S; de Souza, M M; Saraiva, N Z; Tetzner, T A D; Lima, M R; Lopes, F L; Garcia, J M

2012-06-01

Despite extensive efforts, establishment of bovine embryonic stem (ES) cell lines has not been successful. We hypothesized that culture conditions for in vitro-produced (IVP) embryos, the most used source of inner cell mass (ICM) to obtain ES cells, might affect their undifferentiated state. Therefore, the aim of this work was to improve pluripotency of IVP blastocysts to produce suitable ICM for further culturing. We tested KSR and foetal calf serum (FCS) supplements in SOF medium and ES cell conditioned medium (CM) on IVC (groups: KSR, KSR CM, FCS and FCS CM). Cleavage and blastocyst rates were similar between all groups. Also, embryonic quality, assessed by apoptosis rates (TUNEL assay), total cell number and ICM percentage did not differ between experimental groups. However, expression of pluripotency-related markers was affected. We detected down-regulation of OCT3/4, SOX2 and SSEA1 in ICM of FCS CM blastocysts (p < 0.05). SOX2 gene expression revealed lower levels (p < 0.05) on KSR CM blastocysts and a remarkable variation in SOX2 mRNA levels on FCS-supplemented blastocysts. In conclusion, pluripotency-related markers tend to decrease after supplementation with ES cell CM, suggesting different mechanisms regulating mouse and bovine pluripotency. KSR supplementation did not differ from FCS, but FCS replacement by KSR may produce blastocysts with stable SOX2 gene expression levels. © 2011 Blackwell Verlag GmbH.

Validation of the UNC OCT Index for the Diagnosis of Early Glaucoma

PubMed Central

Lee, Gary; Budenz, Donald L.; Warren, Joshua L.; Wall, Michael; Artes, Paul H.; Callan, Thomas M.; Flanagan, John G.

2018-01-01

Purpose To independently validate the performance of the University of North Carolina Optical Coherence Tomography (UNC OCT) Index in diagnosing and predicting early glaucoma. Methods Data of 118 normal subjects (118 eyes) and 96 subjects (96 eyes) with early glaucoma defined as visual field mean deviation (MD) greater than −4 decibels (dB), aged 40 to 80 years, and who were enrolled in the Full-Threshold Testing Size III, V, VI comparison study were used in this study. CIRRUS OCT average and quadrants' retinal nerve fiber layer (RNFL); optic disc vertical cup-to-disc ratio (VCDR), cup-to-disc area ratio, and rim area; and average, minimum, and six sectoral ganglion cell-inner plexiform layer (GCIPL) measurements were run through the UNC OCT Index algorithm. Area under the receiver operating characteristic curve (AUC) and sensitivities at 95% and 99% specificity were calculated and compared between single parameters and the UNC OCT Index. Results Mean age was 60.1 ± 11.0 years for normal subjects and 66.5 ± 8.1 years for glaucoma patients (P < 0.001). MD was 0.29 ± 1.04 dB and −1.30 ± 1.35 dB in normal and glaucomatous eyes (P < 0.001), respectively. The AUC of the UNC OCT Index was 0.96. The best single metrics when compared to the UNC OCT Index were VCDR (0.93, P = 0.054), average RNFL (0.92, P = 0.014), and minimum GCIPL (0.91, P = 0.009). The sensitivities at 95% and 99% specificity were 85.4% and 76.0% (UNC OCT Index), 71.9% and 62.5% (VCDR, all P < 0.001), 64.6% and 53.1% (average RNFL, all P < 0.001), and 66.7% and 58.3% (minimum GCIPL, all P < 0.001), respectively. Conclusions The findings confirm that the UNC OCT Index may provide improved diagnostic perforce over that of single OCT parameters and may be a good tool for detection of early glaucoma. Translational Relevance The UNC OCT Index algorithm may be incorporated easily into routine clinical practice and be useful for detecting early glaucoma. PMID:29629238

Validation of the UNC OCT Index for the Diagnosis of Early Glaucoma.

PubMed

Mwanza, Jean-Claude; Lee, Gary; Budenz, Donald L; Warren, Joshua L; Wall, Michael; Artes, Paul H; Callan, Thomas M; Flanagan, John G

2018-04-01

To independently validate the performance of the University of North Carolina Optical Coherence Tomography (UNC OCT) Index in diagnosing and predicting early glaucoma. Data of 118 normal subjects (118 eyes) and 96 subjects (96 eyes) with early glaucoma defined as visual field mean deviation (MD) greater than -4 decibels (dB), aged 40 to 80 years, and who were enrolled in the Full-Threshold Testing Size III, V, VI comparison study were used in this study. CIRRUS OCT average and quadrants' retinal nerve fiber layer (RNFL); optic disc vertical cup-to-disc ratio (VCDR), cup-to-disc area ratio, and rim area; and average, minimum, and six sectoral ganglion cell-inner plexiform layer (GCIPL) measurements were run through the UNC OCT Index algorithm. Area under the receiver operating characteristic curve (AUC) and sensitivities at 95% and 99% specificity were calculated and compared between single parameters and the UNC OCT Index. Mean age was 60.1 ± 11.0 years for normal subjects and 66.5 ± 8.1 years for glaucoma patients ( P < 0.001). MD was 0.29 ± 1.04 dB and -1.30 ± 1.35 dB in normal and glaucomatous eyes ( P < 0.001), respectively. The AUC of the UNC OCT Index was 0.96. The best single metrics when compared to the UNC OCT Index were VCDR (0.93, P = 0.054), average RNFL (0.92, P = 0.014), and minimum GCIPL (0.91, P = 0.009). The sensitivities at 95% and 99% specificity were 85.4% and 76.0% (UNC OCT Index), 71.9% and 62.5% (VCDR, all P < 0.001), 64.6% and 53.1% (average RNFL, all P < 0.001), and 66.7% and 58.3% (minimum GCIPL, all P < 0.001), respectively. The findings confirm that the UNC OCT Index may provide improved diagnostic perforce over that of single OCT parameters and may be a good tool for detection of early glaucoma. The UNC OCT Index algorithm may be incorporated easily into routine clinical practice and be useful for detecting early glaucoma.

Quantitative mRNA expression analysis of selected genes in patients with early-stage hypothyroidism induced by treatment with iodine-131.

PubMed

Guo, Kun; Gao, Rui; Yu, Yan; Zhang, Weixiao; Yang, Yuxuan; Yang, Aimin

2015-11-01

The present study aimed to investigate the molecular markers indicative of early-stage hypothyroidism induced by treatment with iodine-131, in order to assist in further investigations of radio iodine‑induced hypothyroidism. A total of 59 patients diagnosed with hyperthyroidism (male/female, 16/43; median age, 46.4 years) and 27 healthy subjects (male/female, 7/21; median age, 44.6 years) were included in the present study. All patients were treated with appropriate doses of iodine‑131 and, three months following treatment, the patients were subdivided into two groups: A group with early‑stage hypothyroidism symptoms, and a group with non‑early‑stage hypothyroidism, including euthyroid patients and patients remaining with hyperthyroidism. Tissue samples from the patients and healthy subjects were collected by fine needle biopsies, and the mRNA expression levels of B-cell lymphoma 2 (Bcl‑2), nuclear factor (NF)‑κB, Ku70, epidermal growth factor receptor (EGFR), early growth response 1 (Egr‑1), TP53 and ataxia telangiectasia mutated were analyzed using reverse transcription‑quantitative polymerase chain reaction prior to iodine‑131 treatment. The association of the variation of target genes with susceptibility to early‑stage hypothyroidism was analyzed. Compared with normal subjects, the mRNA expression levels of Ku70 (0.768, vs. 3.304, respectively; P<0.001) and EGFR (0.859, vs. 1.752, respectively; P<0.05) were significantly higher, whereas those of NF‑κB (0.884, vs. 0.578, respectively; P<0.05) and Bcl‑2 (1.235, vs. 0.834, respectively; P<0.05) were lower in the hyperthyroid patients. Following treatment with iodine‑131, 30 of the 59 (50.8%) patients with hyperthyroidism were diagnosed with early‑stage hypothyroidism, and in the early‑stage hypothyroidism group, the mRNA expression levels of Bcl‑2 were significantly decreased (P<0.05), whereas those of Egr‑1 (P<0.05) were significantly increased, compared with the non�

MiR-30e suppresses proliferation of hepatoma cells via targeting prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feng, Guoxing; Shi, Hui; Li, Jiong

Aberrant microRNA expression has been shown to be characteristic of many cancers. It has been reported that the expression levels of miR-30e are decreased in liver cancer tissues. However, the role of miR-30e in hepatocellular carcinoma remains poorly understood. In the present study, we investigated the significance of miR-30e in hepatocarcinogenesis. Bioinformatics analysis reveals a putative target site of miR-30e in the 3′-untranslated region (3′UTR) of prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA. Moreover, luciferase reporter gene assays verified that miR-30e directly targeted 3′UTR of P4HA1 mRNA. Then, we demonstrated that miR-30e was able to reduce the expression of P4HA1 atmore » the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blot analysis. Enforced expression of miR-30e suppressed proliferation of HepG2 cells by 5-ethynyl-2-deoxyuridine (EdU) assay and reduced colony formation of these cells by colony formation analysis. Conversely, anti-miR-30e enhanced the proliferation of hepatoma cells in vitro. Interestingly, the ectopic expression of P4HA1 could efficiently rescue the inhibition of cell proliferation mediated by miR-30e in HepG2 cells. Meanwhile, silencing of P4HA1 abolished the anti-miR-30e-induced proliferation of cells. Clinically, quantitative real-time PCR showed that miR-30e was down-regulated in liver tumor tissues relative to their peritumor tissues. The expression levels of miR-30e were negatively correlated to those of P4HA1 mRNA in clinical liver tumor tissues. Thus, we conclude that miR-30e suppresses proliferation of hepatoma cells through targeting P4HA1 mRNA. Our finding provides new insights into the mechanism of hepatocarcinogenesis. - Highlights: • P4HA1 is a novel target gene of miR-30e. • P4HA1 is increased in clinical HCC tissues. • MiR-30e is negatively correlated with P4HA1 in clinical HCC tissues. • MiR-30e suppresses the proliferation of HCC cells

The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli.

PubMed

Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J; Girbal, Laurence; Cocaign-Bousquet, Muriel

2016-04-26

Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation.

Evaluation of IL-17B and IL-17F mRNA expression in peripheral blood mononuclear cells and association with clinical outcome of IBD patients.

PubMed

Safari, Mohammad Taghi; Chaleshi, Vahid; Tarban, Peyman; Nourian, Mahyar; Balaii, Hedieh; Shahrokh, Shabnam; Asadzadeh Aghdaei, Hamid

2017-01-01

In this study, we determined the gene expression analysis of IL-17 gene family for early detection of subclinical inflammation among IBD patients. Cytokines have a vital role in the pathogenesis of inflammatory bowel disease (IBD). Interleukin-17 is the signature cytokine of the recently identified T helper 17 (Th17) cell subset. IL-17F is mainly involved in mucosal host defense mechanisms whereas the functions of IL-17B remain largely elusive. In this cross-sectional study, IBD patients divided into two active and inactive groups. Peripheral blood mononuclear cells (PBMCs) from 38 IBD patients which 20 inactive samples and 18 active individuals were collected. Changes of IL-17 F and IL-17B mRNA expression level evaluated by quantitative-real time-PCR. mRNA expression level of IL-17B and IL-17F in CD, UC, active and inactive groups have been assessed and there were no significant differences (P>0.05). Patients were classified into five different categories as follows: i) 5ASA; ii) 5ASA + Pred; iii) 5ASA + AZA; iv) 5ASA + Pred + AZA; v) 5ASA + Pred + AZA + IFX according to medication usage, expression of IL-17F and IL-17B had no differences (p>0.05). Evaluation of IL-17B and IL-17F mRNA expression level illustrate no difference among active and inactive patients. Therefore, IL-17B and IL-17F are not biomarkers in an Iranian IBD patients.

Boric acid increases the expression levels of human anion exchanger genes SLC4A2 and SLC4A3.

PubMed

Akbas, F; Aydin, Z

2012-04-03

Boron is an important micronutrient in plants and animals. The role of boron in living systems includes coordinated regulation of gene expression, growth and proliferation of higher plants and animals. There are several well-defined genes associated with boron transportation and tolerance in plants and these genes show close homology with human anion exchanger genes. Mutation of these genes also characterizes some genetic disorders. We investigated the toxic effects of boric acid on HEK293 cells and mRNA expression of anion exchanger (SLC4A1, SLC4A2 and SLC4A3) genes. Cytotoxicity of boric acid at different concentrations was tested by using the methylthiazolyldiphenyl-tetrazolium bromide assay. Gene expression profiles were examined using quantitative real-time PCR. In the HEK293 cells, the nontoxic upper concentration of boric acid was 250 μM; more than 500 μM caused cytotoxicity. The 250 μM boric acid concentration increased gene expression level of SLC4A2 up to 8.6-fold and SLC4A3 up to 2.6-fold, after 36-h incubation. There was no significant effect of boric acid on SLC4A1 mRNA expression levels.

Heterologous expression of the Phycomyces blakesleeanus phytoene dehydrogenase gene (carB) in Mucor circinelloides.

PubMed

Ruiz-Hidalgo, M J; Eslava, A P; Alvarez, M I; Benito, E P

1999-11-01

A phytoene dehydrogenase-deficient mutant of Mucor circinelloides accumulating only phytoene was transformed with the gene encoding the corresponding enzyme (carB gene) of Phycomyces blakesleeanus. Carotenoids derived from phytoene were detected in the transformants showing that the P. blakesleeanus carB gene complements the M. circinelloides carB mutation. These newly formed carotenoids accumulated in low quantities, indicating that functional complementation was poor. carB mRNA molecules correctly transcribed were detected in the transformants, but they represented a small proportion of the total population of carB-derived mRNAs, mostly constituted by truncated transcripts and by transcripts longer than the transcript that is functional in Phycomyces. These results showed that the P. blakesleeanus carB gene was expressed in M. circinelloides and suggested that the poor complementation observed was owing, at least in part, to the lack of specificity in the recognition of the transcription initiation and termination signals of the P. blakesleeanus carB gene by the M. circinelloides transcriptional machinery.

Zaprinast activates MAPKs, NFκB, and Akt and induces the expressions of inflammatory genes in microglia.

PubMed

Lee, Heung-Soon; Kwon, Soon-Ho; Ham, Ji-Eun; Lee, Joo Young; Kim, Dong-Hoon; Shin, Kyung-Ho; Choi, Sang-Hyun

2012-07-01

Previously, the authors reported that zaprinast, an inhibitor of cGMP-selective phosphodiesterases, induced the secretions of TNF-α and IL-1β by microglia and enhanced the induction of iNOS by lipopolysaccharide (LPS). In this study, the signaling mechanism responsible for microglial activation by zaprinast was investigated and the effects of zaprinast and LPS on microglial activation were compared. Zaprinast was found to activate ERK1/2, p38 MAPK, JNK, NFκB, and PI3K/Akt, and subsequently, induce the mRNA expressions of IL-1α, IL-1β, TNF-α, CCL2, CCL4, CXCL1, CXCL2, and CD14. Associations between signaling pathways and gene expressions were examined by treating microglia with signal inhibitors. PDTC inhibited the induction of all the above genes by zaprinast, and SB203580 inhibited all genes except CXCL1. SP600125, PD98059, and LY294002 inhibited the induction of at least CCL2. Microglial activation by zaprinast was then compared with full-blown activation by LPS. The zaprinast-induced phosphorylations of MAPKs and IκB were less prompt than LPS-induced phosphorylations. IκB degradation by LPS was significant at 10min and did not return to normal, whereas zaprinast induced a later, transient degradation. LPS induced the mRNA expressions of IL-1β, TNF-α, IL-6, CCL2, iNOS, and COX-2, and although zaprinast significantly induced the expressions of all except IL-6 and iNOS, these inductions were far less than those induced by LPS. Collectively, zaprinast was found to upregulate microglial activity mainly via NFκB and p38 MAPK signaling and the subsequent expressions of inflammatory genes. Although, zaprinast was found to have obvious effects on microglia, these were weaker than the effects of LPS. Copyright © 2012 Elsevier B.V. All rights reserved.

FOXP3, CBLB and ITCH gene expression and cytotoxic T lymphocyte antigen 4 expression on CD4+CD25high T cells in multiple sclerosis

PubMed Central

Sellebjerg, F; Krakauer, M; Khademi, M; Olsson, T; Sørensen, P S

2012-01-01

Expression of the forkhead box protein 3 (FoxP3) transcription factor is regulated by the E3 ubiquitin ligases Itch and Cbl-b and induces regulatory activity CD4+CD25high T cells. Treatment with interferon (IFN)-β enhances regulatory T cell activity in multiple sclerosis (MS). We studied the phenotype of CD4+CD25high T cells in MS by flow cytometry and its relationship with expression of the FOXP3, ITCH and CBLB genes. We found that untreated MS patients had lower cell surface expression of cytotoxic T lymphocyte antigen 4 (CTLA-4) on CD4+CD25high T cells and higher intracellular CTLA-4 expression than healthy controls. Cell surface expression of CTLA-4 on CD4+CD25high T cells correlated with expression of FOXP3 mRNA in untreated patients and increased significantly with time from most recent injection in patients treated with IFN-β. FOXP3 mRNA expression correlated with CBLB and ITCH and T helper type 2 cytokine mRNA expression in MS patients. These data link expression of FOXP3, CBLB and ITCH mRNA and CTLA-4 expression on the surface of CD4+CD25high T cell in MS. We hypothesize that this may reflect alterations in the inhibitory effect of CTLA-4 or in regulatory T cell function. PMID:23039885

Hepatitis B surface antigen gene expression is regulated by sex steroids and glucocorticoids in transgenic mice.

PubMed Central

Farza, H; Salmon, A M; Hadchouel, M; Moreau, J L; Babinet, C; Tiollais, P; Pourcel, C

1987-01-01

We have investigated the basis for liver-specific and sex-linked expression of hepatitis B surface antigen (HBsAg) gene in transgenic mice by monitoring the level of liver HBsAg mRNA and serum HBsAg at different stages of development and in response to sex-hormone regulation. Transcription of the HBsAg gene starts at day 15 of development, together with that of the albumin gene, and reaches a comparable level at birth. HBsAg mRNA level and HBsAg production are parallel in males and females during prenatal development and until the first month of life, but HBsAg gene expression increases 5-10 times in males at puberty. After castration, the level of expression decreases dramatically in both males and females and is subsequently increased by injection of testosterone or estradiol. Glucocorticoids also regulated positively expression of the HBsAg gene. Our results suggest that sex hormones play a role in hepatitis B virus gene expression during natural infection and could explain the difference in incidence of chronic carriers between men and women. Images PMID:3469661

B-cell transcription factors Pax-5, Oct-2, BOB.1, Bcl-6, and MUM1 are useful markers for the diagnosis of nodular lymphocyte predominant Hodgkin lymphoma.

PubMed

Herbeck, Rosemarie; Teodorescu Brînzeu, D; Giubelan, Marioara; Lazăr, Elena; Dema, Alis; Ioniţă, Hortensia

2011-01-01

In some instances, the overlap in morphologic features and antigen expression between nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) and classical Hodgkin lymphoma (cHL) can cause confusion in the diagnosis. In these cases, the transcription factors (TFs) B-cell specific activator protein (BSAP)/Pax-5, octamer binding protein-2 (Oct-2), B-lymphocyte-specific co-activator BOB.1/OBF.1, Bcl-6 protein and multiple myeloma-1/interferon regulatory factor-4 (MUM1/IRF-4) may aid in clarifying the diagnosis. Twenty-two cases of NLPHL were studied for the immunohistochemical expression of Pax-5, Oct-2, BOB.1, Bcl-6 protein and MUM1/IRF-4. Our results sustain the usefulness of the selected set of TFs to diagnose and distinguish NLPHL from cHL since Pax-5, Oct-2, BOB.1 and Bcl-6 are consistently expressed by lymphocyte predominant (LP) cells and reported by others to be often unexpressed in Hodgkin and Reed-Sternberg cells. By contrast, MUM1/IRF-4 protein scored negative in the majority of LP cells, but is reported to be expressed in almost all cases of cHL. Thus, although the expression of transcription factors is very heterogeneous, their simultaneous implementation for positive and differential diagnosis may be useful.

In situ Localization of the Human Multidrug‐resistance Gene mRNA Using Thymine‐Thymine Dimerized Single‐stranded cDNA

PubMed Central

Koji, Takehiko; Ueda, Kazumitsu; Pastan, Ira; Gottesman, Michael M.; Nakane, Paul K.; Mori, Shigeo

1990-01-01

In order to detect the mRNA transcribed from the multidrug‐resistance gene (MDR1), thymine‐thymine (T‐T) dimerized single‐stranded DNA probes have been utilized for hybridization with mRNA either on nitrocellulose filters or in cells and tissues. S1 nuclease digestion rather than sonication was used to obtain short T‐T dimerized single‐stranded DNA (300–400 bases) so that they could penetrate well into the cytoplasm. The hybridized T‐T DNA was detected immunohisto‐chemically using rabbit anti‐T‐T DNA antibody (Ab) and peroxidase‐labeled goat anti‐rabbit IgG Ab. Employing this system, MDR1 mRNA could be localized clearly in the human multidrug‐resistant cell lines K562/ADM, CEM/VLB, 2780ad, and KBC4 cells as well as in human fetal kidney and gastric carcinoma. Furthermore, our system successfully detected the expression of MDR1 mRNA in cell lines of increasing resistance. These results paralleled results obtained at the protein level by immunohistochemistry. The analysis of MDR1 RNA expression by this in situ hybridization technique should be useful in the study of normal human tissues and tumor samples expressing the MDR1 gene. PMID:1977730

Visualization and tissue classification of human breast cancer images using ultrahigh-resolution OCT.

PubMed

Yao, Xinwen; Gan, Yu; Chang, Ernest; Hibshoosh, Hanina; Feldman, Sheldon; Hendon, Christine

2017-03-01

Breast cancer is one of the most common cancers, and recognized as the third leading cause of mortality in women. Optical coherence tomography (OCT) enables three dimensional visualization of biological tissue with micrometer level resolution at high speed, and can play an important role in early diagnosis and treatment guidance of breast cancer. In particular, ultra-high resolution (UHR) OCT provides images with better histological correlation. This paper compared UHR OCT performance with standard OCT in breast cancer imaging qualitatively and quantitatively. Automatic tissue classification algorithms were used to automatically detect invasive ductal carcinoma in ex vivo human breast tissue. Human breast tissues, including non-neoplastic/normal tissues from breast reduction and tumor samples from mastectomy specimens, were excised from patients at Columbia University Medical Center. The tissue specimens were imaged by two spectral domain OCT systems at different wavelengths: a home-built ultra-high resolution (UHR) OCT system at 800 nm (measured as 2.72 μm axial and 5.52 μm lateral) and a commercial OCT system at 1,300 nm with standard resolution (measured as 6.5 μm axial and 15 μm lateral), and their imaging performances were analyzed qualitatively. Using regional features derived from OCT images produced by the two systems, we developed an automated classification algorithm based on relevance vector machine (RVM) to differentiate hollow-structured adipose tissue against solid tissue. We further developed B-scan based features for RVM to classify invasive ductal carcinoma (IDC) against normal fibrous stroma tissue among OCT datasets produced by the two systems. For adipose classification, 32 UHR OCT B-scans from 9 normal specimens, and 28 standard OCT B-scans from 6 normal and 4 IDC specimens were employed. For IDC classification, 152 UHR OCT B-scans from 6 normal and 13 IDC specimens, and 104 standard OCT B-scans from 5 normal and 8 IDC specimens

STAT5A and STAT5B have opposite correlations with drug response gene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lamba, V., E-mail: vlamba@ufl.edu; Jia, B.; Liang, F.

Introduction: STAT5A and STAT5B are important transcription factors that play a key role in regulation of several important physiological processes including proliferation, survival, mediation of responses to cytokines and in regulating gender differences in drug response genes such as the hepatic cytochrome P450s (CYPs) that are responsible for a large majority of drug metabolism reactions in the human body. STAT5A and STAT5b have a high degree of sequence homology and have been reported to have largely similar functions. Recent studies have, however, indicated that they can also often have distinct and unique roles in regulating gene expression. Objective: In thismore » study, we evaluated the association of STAT5A and STAT5B mRNA expression levels with those of several key hepatic cytochrome P450s (CYPs) and hepatic transcription factors (TFs) and evaluated the potential roles of STAT5A and 5b in mediating gender differences in these CYPs and TFs. Methods: Expression profiling for major hepatic CYP isoforms and transcription factors was performed using RNA sequencing (RNA-seq) in 102 human liver samples (57 female, 45 male). Real time PCR gene expression data for selected CYPs and TFs was available on a subset of 50 human liver samples (25 female, 25 male) and was used to validate the RNA-seq findings. Results: While STAT5A demonstrated significant negative correlation with expression levels of multiple hepatic transcription factors (including NR1I2 and HNF4A) and DMEs such as CYP3A4 and CYP2C19, STAT5B expression was observed to demonstrate positive associations with several CYPs and TFs analyzed. As STAT5A and STAT5B have been shown to be important in regulation of gender differences in CYPs, we also analyzed STAT5A and 5b associations with CYPs and TFs separately in males and females and observed gender dependent differential associations of STATs with several CYPs and TFs. Results from the real time PCR validation largely supported our RNA-seq findings

Altered profile of mRNA expression in atrioventricular node of streptozotocin-induced diabetic rats

PubMed Central

Howarth, Frank Christopher; Parekh, Khatija; Jayaprakash, Petrilla; Inbaraj, Edward Samuel; Oz, Murat; Dobrzynski, Halina; Adrian, Thomas Edward

2017-01-01

Prolonged action potential duration, reduced action potential firing rate, upstroke velocity and rate of diastolic depolarization have been demonstrated in atrioventricular node (AVN) cells from streptozotocin (STZ)-induced diabetic rats. To further clarify the molecular basis of these electrical disturbances, the mRNA profiles encoding a variety of proteins associated with the generation and conduction of electrical activity in the AVN, were evaluated in the STZ-induced diabetic rat heart. Expression of mRNA was measured in AVN biopsies using reverse transcription-quantitative polymerase chain reaction techniques. Notable differences in mRNA expression included upregulation of genes encoding membrane and intracellular Ca2+ transport, including solute carrier family 8 member A1, transient receptor potential channel 1, ryanodine receptor 2/3, hyperpolarization-activated cyclic-nucleotide 2 and 3, calcium channel voltage-dependent, β2 subunit and sodium channels 3a, 4a, 7a and 3b. In addition to this, potassium channels potassium voltage-gated channel subfamily A member 4, potassium channel calcium activated intermediate/small conductance subfamily N α member 2, potassium voltage-gated channel subfamily J members 3, 5, and 11, potassium channel subfamily K members 1, 2, 3 and natriuretic peptide B (BNP) were upregulated in AVN of STZ heart, compared with controls. Alterations in gene expression were associated with upregulation of various proteins including the inwardly rectifying, potassium channel Kir3.4, NCX1 and BNP. The present study demonstrated notable differences in the profile of mRNA encoding proteins associated with the generation, conduction and regulation of electrical signals in the AVN of the STZ-induced diabetic rat heart. These data will provide a basis for a substantial range of future studies to investigate whether variations in mRNA translate into alterations in electrophysiological function. PMID:28731153

Immunohistochemical analysis of the role and relationship between Notch-1 and Oct-4 expression in urinary bladder carcinoma.

PubMed

Abdou, Asmaa Gaber; El-Wahed, Moshira Mohammed Abd; Kandil, Mona Abd-Elhalim; Samaka, Rehab Monir; Elkady, Noha

2013-10-01

Most tumors contain a minor population of cancer stem cells that are responsible for tumor heterogeneity, resistance to therapy and recurrence. Oct-4 is a transcription factor responsible for self-renewal of stem cells, whereas the Notch family of receptors and ligands may play a pivotal role in the regulation of stem cell maintenance and differentiation. This study aimed at an evaluation of Oct-4 and Notch-1 expression in both carcinoma and stromal cells of 83 cases of primary bladder carcinoma and to study the relationship between them. Notch-1 was expressed in carcinoma and stromal cells of all malignant cases, where expression in both cell types was correlated with parameters indicating differentiation, such as low grade (p < 0.05) and less proliferation (p < 0.05). However, Notch-1 expression in stromal cells was associated with nodal metastasis (p = 0.016) and advanced stage (p = 0.030). 56.6 and 75.9% of carcinoma and stromal cells of malignant cases showed Oct-4 expression, respectively. Oct-4 expression in carcinoma cells or stromal cells was associated with aggressive features of bladder carcinoma, such as poor differentiation (p = 0.001), high proliferation (p < 0.001, 0.030), and liability for recurrence (p = 0.010, p < 0.001). There was an inverse relationship between Notch-1 and Oct-4 expression in carcinoma cells (p = 0.002), but stromal expression of Notch-1 was found to be associated with a nuclear pattern of Oct-4 expression in carcinoma cells (p = 0.030). Oct-4 as a stem cell marker is expressed in carcinoma cells and in stromal cells of bladder carcinoma, where they may cooperate in the progression of bladder carcinoma by acquiring aggressive features, such as a liability for recurrence and dissemination. Notch-1 is also expressed in both carcinoma cells and stromal cells of bladder carcinoma. Although they could share in enhancing differentiation, stromal expression of Notch-1 may have a bad impact, possibly through up-regulation of the active

Identification of Powdery Mildew Responsive Genes in Hevea brasiliensis through mRNA Differential Display

PubMed Central

Li, Xiang; Bi, Zhenghong; Di, Rong; Liang, Peng; He, Qiguang; Liu, Wenbo; Miao, Weiguo; Zheng, Fucong

2016-01-01

Powdery mildew is an important disease of rubber trees caused by Oidium heveae B. A. Steinmann. As far as we know, none of the resistance genes related to powdery mildew have been isolated from the rubber tree. There is little information available at the molecular level regarding how a rubber tree develops defense mechanisms against this pathogen. We have studied rubber tree mRNA transcripts from the resistant RRIC52 cultivar by differential display analysis. Leaves inoculated with the spores of O. heveae were collected from 0 to 120 hpi in order to identify pathogen-regulated genes at different infection stages. We identified 78 rubber tree genes that were differentially expressed during the plant–pathogen interaction. BLAST analysis for these 78 ESTs classified them into seven functional groups: cell wall and membrane pathways, transcription factor and regulatory proteins, transporters, signal transduction, phytoalexin biosynthesis, other metabolism functions, and unknown functions. The gene expression for eight of these genes was validated by qRT-PCR in both RRIC52 and the partially susceptible Reyan 7-33-97 cultivars, revealing the similar or differential changes of gene expressions between these two cultivars. This study has improved our overall understanding of the molecular mechanisms of rubber tree resistance to powdery mildew. PMID:26840302

CCR4-NOT deadenylates mRNA associated with RNA-induced silencing complexes in human cells.

PubMed

Piao, Xianghua; Zhang, Xue; Wu, Ligang; Belasco, Joel G

2010-03-01

MicroRNAs (miRNAs) repress gene expression posttranscriptionally by inhibiting translation and by expediting deadenylation so as to trigger rapid mRNA decay. Their regulatory influence is mediated by the protein components of the RNA-induced silencing complex (RISC), which deliver miRNAs and siRNAs to their mRNA targets. Here, we present evidence that CCR4-NOT is the deadenylase that removes poly(A) from messages destabilized by miRNAs in human cells. Overproducing a mutationally inactivated form of either of the catalytic subunits of this deadenylase (CCR4 or CAF1/POP2) significantly impedes the deadenylation and decay of mRNA targeted by a partially complementary miRNA. The same deadenylase initiates the degradation of "off-target" mRNAs that are bound by an imperfectly complementary siRNA introduced by transfection. The greater inhibitory effect of inactive CAF1 or POP2 (versus inactive CCR4) suggests a predominant role for this catalytic subunit of CCR4-NOT in miRNA- or small interfering RNA (siRNA)-mediated deadenylation. These effects of mi/siRNAs and CCR4-NOT can be fully reproduced by directly tethering RISC to mRNA without the guidance of a small RNA, indicating that the ability of RISC to accelerate deadenylation is independent of RNA base pairing. Despite its importance for mi/siRNA-mediated deadenylation, CCR4-NOT appears not to associate significantly with RISC, as judged by the failure of CAF1 and POP2 to coimmunoprecipitate detectably with either the Ago or TNRC6 subunit of RISC, a finding at odds with deadenylase recruitment as the mechanism by which RISC accelerates poly(A) removal.

Ethanol-induced changes in Poly (ADP ribose) Polymerase and neuronal developmental gene expression

PubMed Central

Gavin, David P.; Kusumo, Handojo; Sharma, Rajiv P.; Guizzetti, Marina

2016-01-01

Prenatal alcohol exposure has profound effects on neuronal growth and development. Poly-ADP Ribose Polymerase (PARP) enzymes are perhaps unique in the field of epigenetics in that they directly participate in histone modifications, transcription factor modifications, DNA methylation/demethylation and are highly inducible by ethanol. It was our hypothesis that ethanol would induce PARP enzymatic activity leading to alterations in neurodevelopmental gene expression. Mouse E18 cortical neurons were treated with ethanol, PARP inhibitors, and nuclear hormone receptor transcription factor PPARγ agonists and antagonists. Subsequently, we measured PARP activity and changes in Bdnf, OKSM (Oct4, Klf4, Sox2, c-Myc), DNA methylating/demethylating factors, and Pparγ mRNA expression, promoter 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC), and PPARγ promoter binding. We found that ethanol reduced Bdnf4, 9a, and Klf4 mRNA expression, and increased c-Myc expression. These changes were reversed with a PARP inhibitor. In agreement with its role in DNA demethylation PARP inhibition increased 5MC levels at the c-Myc promoter. In addition, we found that elevated PARP enzymatic activity reduced PPARγ promoter binding, and this corresponded to decreased Bdnf and Klf4 mRNA expression. Our results suggest that PARP participates in DNA demethylation and reduces PPARγ promoter binding. The current study underscores the importance of PARP in ethanol-induced changes to neurodevelopmental gene expression. PMID:27497606

Over-expression of Oct4 and Sox2 transcription factors enhances differentiation of human umbilical cord blood cells in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guseva, Daria; Hannover Medical School, Hannover; Rizvanov, Albert A.

2014-09-05

Highlights: • Gene and cell-based therapies comprise innovative aspects of regenerative medicine. • Genetically modified hUCB-MCs enhanced differentiation of cells in a mouse model of ALS. • Stem cells successfully transformed into micro-glial and endothelial lines in spinal cords. • Over-expressing oct4 and sox2 also induced production of neural marker PGP9.5. • Formation of new nerve cells, secreting trophic factors and neo-vascularisation could improve symptoms in ALS. - Abstract: Gene and cell-based therapies comprise innovative aspects of regenerative medicine. Even though stem cells represent a highly potential therapeutic strategy, their wide-spread exploitation is marred by ethical concerns, potential for malignantmore » transformation and a plethora of other technical issues, largely restricting their use to experimental studies. Utilizing genetically modified human umbilical cord blood mono-nuclear cells (hUCB-MCs), this communication reports enhanced differentiation of transplants in a mouse model of amyotrophic lateral sclerosis (ALS). Over-expressing Oct4 and Sox2 induced production of neural marker PGP9.5, as well as transformation of hUCB-MCs into micro-glial and endothelial lines in ALS spinal cords. In addition to producing new nerve cells, providing degenerated areas with trophic factors and neo-vascularisation might prevent and even reverse progressive loss of moto-neurons and skeletal muscle paralysis.« less

Expression of activation-induced cytidine deaminase gene in B lymphocytes of patients with common variable immunodeficiency.

PubMed

Abolhassani, Hassan; Farrokhi, Amir Salek; Pourhamdi, Shabnam; Mohammadinejad, Payam; Sadeghi, Bamdad; Moazzeni, Seyed-Mohammad; Aghamohammadi, Asghar

2013-08-01

Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by reduced serum level of IgG, IgA or IgM and recurrent bacterial infections. Class switch recombination (CSR) as a critical process in immunoglobulin production is defective in a group of CVID patients. Activation-induced cytidine deaminase (AID) protein is an important molecule involving CSR process. The aim of this study was to investigate the AID gene mRNA production in a group of CVID patients indicating possible role of this molecule in this disorder. Peripheral blood mononuclear cells (PBMC) of 29 CVID patients and 21 healthy controls were isolated and stimulated by CD40L and IL-4 to induce AID gene expression. After 5 days AID gene mRNA production was investigated by real time polymerase chain reaction. AID gene was expressed in all of the studied patients. However the mean density of extracted AID mRNA showed higher level in CVID patients (230.95±103.04 ng/ml) rather than controls (210.00±44.72 ng/ml; P=0.5). CVID cases with lower level of AID had decreased total level of IgE (P=0.04) and stimulated IgE production (P=0.02); while cases with increased level of AID presented higher level of IgA (P=0.04) and numbers of B cells (P=0.02) and autoimmune disease (P=0.02). Different levels of AID gene expression may have important roles in dysregulation of immune system and final clinical presentation in CVID patients. Therefore investigating the expression of AID gene can help in classifying CVID patients.

Association of differential β-catenin expression with Oct-4 and Nanog in oral squamous cell carcinoma and their correlation with clinicopathological factors and prognosis.

PubMed

Ravindran, Gokulan; Sawant, Sharada S; Hague, Angela; Kingsley, Karl; Devaraj, Halagowder

2015-07-01

The re-expression of pluripotent markers (Oct-4 and Nanog) and the reactivation of stem cell-related pathways in oral carcinoma have been well researched. However, the relationship between the stem cell signaling molecule β-catenin and pluripotent markers Oct-4 and Nanog in oral cancer is yet to be studied in detail. Therefore, we have investigated the correlation among Oct-4, Nanog, and β-catenin in oral squamous cell carcinoma, which, in turn, could provide valuable insight into its prognostic significance. The immunohistochemical analysis was performed for 60 cases of oral cancer to study the expression pattern of Oct-4, Nanog, and β-catenin. Whereas immunofluorescence analysis was used to investigate the co-localization of β-catenin with Oct-4 and Nanog in oral carcinoma tissues and H314 cell line. Finally, co-immunoprecipitation analysis was used to study the possible interaction between β-catenin and Oct-4 in oral carcinoma cells. β-catenin, Oct-4, and Nanog showed significant correlation with lymph node metastasis, stage, grade, and prognosis in oral squamous cell carcinoma. Interestingly, a significant positive correlation was found among the expression of Oct-4, Nanog, and β-catenin. Moreover, the interaction between β-catenin and Oct-4 was observed in oral cancer. The positive correlation among Oct-4, Nanog, and β-catenin suggests their coordinated role in maintaining proliferation in oral carcinoma cells. The interaction between β-catenin and Oct-4 may be a crucial event in oral carcinogenesis. On the other hand, β-catenin, Oct-4, and Nanog could be used as independent prognostic markers of oral squamous cell carcinoma. © 2014 Wiley Periodicals, Inc.

A selection of reference genes and early-warning mRNA biomarkers for environmental monitoring using Mytilus spp. as sentinel species.

PubMed

Lacroix, C; Coquillé, V; Guyomarch, J; Auffret, M; Moraga, D

2014-09-15

mRNA biomarkers are promising tools for environmental health assessment and reference genes are needed to perform relevant qPCR analyses in tissue samples of sentinel species. In the present study, potential reference genes and mRNA biomarkers were tested in the gills and digestive glands of native and caged mussels (Mytilus spp.) exposed to harbor pollution. Results highlighted the difficulty to find stable reference genes in wild, non-model species and suggested the use of normalization indices instead of single genes as they exhibit a higher stability. Several target genes were found differentially expressed between mussel groups, especially in gills where cyp32, π-gst and CuZn-sod mRNA levels could be biomarker candidates. Multivariate analyses confirmed the ability of mRNA levels to highlight site-effects and suggested the use of several combined markers instead of individual ones. These findings support the use of qPCR technology and mRNA levels as early-warning biomarkers in marine monitoring programs. Copyright © 2014 Elsevier Ltd. All rights reserved.

Knockdown of stem cell regulator Oct4A in ovarian cancer reveals cellular reprogramming associated with key regulators of cytoskeleton-extracellular matrix remodelling

PubMed Central

Samardzija, Chantel; Greening, David W.; Escalona, Ruth; Chen, Maoshan; Bilandzic, Maree; Luwor, Rodney; Kannourakis, George; Findlay, Jock K.; Ahmed, Nuzhat

2017-01-01

Oct4A is a master regulator of self-renewal and pluripotency in embryonic stem cells. It is a well-established marker for cancer stem cell (CSC) in malignancies. Recently, using a loss of function studies, we have demonstrated key roles for Oct4A in tumor cell survival, metastasis and chemoresistance in in vitro and in vivo models of ovarian cancer. In an effort to understand the regulatory role of Oct4A in tumor biology, we employed the use of an ovarian cancer shRNA Oct4A knockdown cell line (HEY Oct4A KD) and a global mass spectrometry (MS)-based proteomic analysis to investigate novel biological targets of Oct4A in HEY samples (cell lysates, secretomes and mouse tumor xenografts). Based on significant differential expression, pathway and protein network analyses, and comprehensive literature search we identified key proteins involved with biologically relevant functions of Oct4A in tumor biology. Across all preparations of HEY Oct4A KD samples significant alterations in protein networks associated with cytoskeleton, extracellular matrix (ECM), proliferation, adhesion, metabolism, epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) and drug resistance was observed. This comprehensive proteomics study for the first time presents the Oct4A associated proteome and expands our understanding on the biological role of this stem cell regulator in carcinomas. PMID:28406185

Sodium 4-phenylbutyrate downregulates HSC70 expression by facilitating mRNA degradation.

PubMed

Rubenstein, R C; Lyons, B M

2001-07-01

Intracellular trafficking of the DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) is repaired by sodium 4-phenylbutyrate (4PBA) by an undetermined mechanism. 4PBA downregulates protein and mRNA expression of the heat shock cognate protein HSC70 (the constitutively expressed member of the 70-kDa heat shock protein family) by approximately 40-50% and decreases formation of a HSC70-DeltaF508 CFTR complex that may be important in the intracellular degradation of DeltaF508 CFTR. We examined the potential mechanisms by which 4PBA decreases HSC70 mRNA and protein expression. In IB3-1 cells, 1 mM 4PBA did not alter the activity of the Chinese hamster ovary HSC70 promoter or of a human HSC70 promoter fragment in luciferase reporter assays nor did it alter HSC70 mRNA synthesis in nuclear runoff assays. In contrast, preincubation with 4PBA increased the rate of HSC70 mRNA degradation by approximately 40%. The initial rate of 35S-HSC70 protein synthesis in 4PBA-treated IB3-1 cells was reduced by approximately 40%, consistent with the steady-state mRNA level, whereas its rate of degradation was unaltered by 4PBA. 4PBA also reduced the steady-state accumulation of (35)S-HSC70 by approximately 40%. These data suggest that 4PBA decreases the expression of HSC70 mRNA and protein by inducing cellular adaptations that result in the decreased stability of HSC70 mRNA.

Anesthesia for Euthanasia Influences mRNA Expression in Healthy Mice and after Traumatic Brain Injury

PubMed Central

Staib-Lasarzik, Irina; Kriege, Oliver; Timaru-Kast, Ralph; Pieter, Dana; Werner, Christian; Engelhard, Kristin

2014-01-01

Abstract Tissue sampling for gene expression analysis is usually performed under general anesthesia. Anesthetics are known to modulate hemodynamics, receptor-mediated signaling cascades, and outcome parameters. The present study determined the influence of anesthetic paradigms typically used for euthanization and tissue sampling on cerebral mRNA expression in mice. Naïve mice and animals with acute traumatic brain injury induced by controlled cortical impact (CCI) were randomized to the following euthanasia protocols (n=10–11/group): no anesthesia (NA), 1 min of 4 vol% isoflurane in room air (ISO), 3 min of a combination of 5 mg/kg midazolam, 0.05 mg/kg fentanyl, and 0.5 mg/kg medetomidine intraperitoneally (COMB), or 3 min of 360 mg/kg chloral hydrate intraperitoneally (CH). mRNA expression of actin-1-related gene (Act1), FBJ murine osteosarcoma viral oncogene homolog B (FosB), tumor necrosis factor alpha (TNFα), heat shock protein beta-1 (HspB1), interleukin (IL)-6, tight junction protein 1 (ZO-1), IL-1ß, cyclophilin A, micro RNA 497 (miR497), and small cajal body-specific RNA 17 were determined by real-time polymerase chain reaction (PCR) in hippocampus samples. In naïve animals, Act1 expression was downregulated in the CH group compared with NA. FosB expression was downregulated in COMB and CH groups compared with NA. CCI reduced Act1 and FosB expression, whereas HspB1 and TNFα expression increased. After CCI, HspB1 expression was significantly higher in ISO, COMB, and CH groups, and TNFα expression was elevated in ISO and COMB groups. MiR497, IL-6, and IL-1ß were upregulated after CCI but not affected by anesthetics. Effects were independent of absolute mRNA copy numbers. The data demonstrate that a few minutes of anesthesia before tissue sampling are sufficient to induce immediate mRNA changes, which seem to predominate in the early-regulated gene cluster. Anesthesia-related effects on gene expression might explain limited

Anesthesia for euthanasia influences mRNA expression in healthy mice and after traumatic brain injury.

PubMed

Staib-Lasarzik, Irina; Kriege, Oliver; Timaru-Kast, Ralph; Pieter, Dana; Werner, Christian; Engelhard, Kristin; Thal, Serge C

2014-10-01

Tissue sampling for gene expression analysis is usually performed under general anesthesia. Anesthetics are known to modulate hemodynamics, receptor-mediated signaling cascades, and outcome parameters. The present study determined the influence of anesthetic paradigms typically used for euthanization and tissue sampling on cerebral mRNA expression in mice. Naïve mice and animals with acute traumatic brain injury induced by controlled cortical impact (CCI) were randomized to the following euthanasia protocols (n=10-11/group): no anesthesia (NA), 1 min of 4 vol% isoflurane in room air (ISO), 3 min of a combination of 5 mg/kg midazolam, 0.05 mg/kg fentanyl, and 0.5 mg/kg medetomidine intraperitoneally (COMB), or 3 min of 360 mg/kg chloral hydrate intraperitoneally (CH). mRNA expression of actin-1-related gene (Act1), FBJ murine osteosarcoma viral oncogene homolog B (FosB), tumor necrosis factor alpha (TNFα), heat shock protein beta-1 (HspB1), interleukin (IL)-6, tight junction protein 1 (ZO-1), IL-1ß, cyclophilin A, micro RNA 497 (miR497), and small cajal body-specific RNA 17 were determined by real-time polymerase chain reaction (PCR) in hippocampus samples. In naïve animals, Act1 expression was downregulated in the CH group compared with NA. FosB expression was downregulated in COMB and CH groups compared with NA. CCI reduced Act1 and FosB expression, whereas HspB1 and TNFα expression increased. After CCI, HspB1 expression was significantly higher in ISO, COMB, and CH groups, and TNFα expression was elevated in ISO and COMB groups. MiR497, IL-6, and IL-1ß were upregulated after CCI but not affected by anesthetics. Effects were independent of absolute mRNA copy numbers. The data demonstrate that a few minutes of anesthesia before tissue sampling are sufficient to induce immediate mRNA changes, which seem to predominate in the early-regulated gene cluster. Anesthesia-related effects on gene expression might explain limited reproduciblity of real

RNA editing and regulation of Drosophila 4f-rnp expression by sas-10 antisense readthrough mRNA transcripts

PubMed Central

PETERS, NICK T.; ROHRBACH, JUSTIN A.; ZALEWSKI, BRIAN A.; BYRKETT, COLLEEN M.; VAUGHN, JACK C.

2003-01-01

We have previously described an example of extensively A-to-G edited cDNA derived from adult heads of the fruitfly Drosophila melanogaster. In that study, the source of the predicted antisense RNA pairing strand for template recognition by dADAR editase was not identified, and the biological significance of the observed hyperediting was not known. Here, we address each of these questions. 4f-rnp and sas-10 are closely adjacent X-linked genes located on opposite DNA strands that produce convergent transcripts. We show that developmentally regulated antisense sas-10 readthrough mRNA arises by activation of an upstream promoter P2 during the late embryo stage of fly development. The sas-10 readthrough transcripts pair with 4f-rnp mRNA to form double-stranded molecules, as indicated by A-to-G editing observed in both RNA strands. It would be predicted that perfect RNA duplexes would be targeted for modification/degradation by enzyme pathways that recognize double-stranded RNAs, leading to decline in 4f-rnp mRNA levels, and this is what we observe. The observation using quantitative RT-PCR that sas-10 readthrough and 4f-rnp transcript levels are inversely related suggests a role for the antisense RNA in posttranscriptional regulation of 4f-rnp gene expression during development. Potential molecular mechanisms that could lead to this result are discussed, one of which is targeted transcript degradation via the RNAi pathway. Insofar as the dADAR editase and RNAi pathways are known to be constitutive in this system, it is likely that control of antisense RNA transcription is the rate-limiting factor. The results provide insight into roles of naturally occurring antisense RNAs in regulation of eukaryotic gene expression. PMID:12756328

Evaluation of IL-17B and IL-17F mRNA expression in peripheral blood mononuclear cells and association with clinical outcome of IBD patients

PubMed Central

Safari, Mohammad Taghi; Chaleshi, Vahid; Tarban, Peyman; Nourian, Mahyar; Balaii, Hedieh; Shahrokh, Shabnam; Asadzadeh Aghdaei, Hamid

2017-01-01

Aim: In this study, we determined the gene expression analysis of IL-17 gene family for early detection of subclinical inflammation among IBD patients. Background: Cytokines have a vital role in the pathogenesis of inflammatory bowel disease (IBD). Interleukin-17 is the signature cytokine of the recently identified T helper 17 (Th17) cell subset. IL-17F is mainly involved in mucosal host defense mechanisms whereas the functions of IL-17B remain largely elusive. Methods: In this cross-sectional study, IBD patients divided into two active and inactive groups. Peripheral blood mononuclear cells (PBMCs) from 38 IBD patients which 20 inactive samples and 18 active individuals were collected. Changes of IL-17 F and IL-17B mRNA expression level evaluated by quantitative-real time-PCR. Results: mRNA expression level of IL-17B and IL-17F in CD, UC, active and inactive groups have been assessed and there were no significant differences (P>0.05). Patients were classified into five different categories as follows: i) 5ASA; ii) 5ASA + Pred; iii) 5ASA + AZA; iv) 5ASA + Pred + AZA; v) 5ASA + Pred + AZA + IFX according to medication usage, expression of IL-17F and IL-17B had no differences (p>0.05). Conclusion: Evaluation of IL-17B and IL-17F mRNA expression level illustrate no difference among active and inactive patients. Therefore, IL-17B and IL-17F are not biomarkers in an Iranian IBD patients. PMID:29511476

Micro-tattoo guided OCT imaging of site specific inflammation

NASA Astrophysics Data System (ADS)

Phillips, Kevin G.; Choudhury, Niloy; Samatham, Ravikant V.; Singh, Harvinder; Jacques, Steven L.

2010-02-01

Epithelial biologists studying human skin diseases such as cancer formation and psoriasis commonly utilize mouse models to characterize the interplay among cells and intracellular signal transduction pathways that result in programmed changes in gene expression and cellular behaviors. The information obtained from animal models is useful only when phenotypic presentations of disease recapitulate those observed in humans. Excision of tissues followed by histochemical analysis is currently the primary means of establishing the morphological presentation. Non invasive imaging of animal models provides an alternate means to characterize tissue morphology associated with the disease of interest in vivo. While useful, the ability to perform in vivo imaging at different time points in the same tissue location has been a challenge. This information is key to understanding site specific changes as the imaged tissue can now be extracted and analyzed for mRNA expression. We present a method employing a micro-tattoo to guide optical coherence tomography (OCT) imaging of ultraviolet induced inflammation over time in the same tissue locations.

Scalable topographies to support proliferation and Oct4 expression by human induced pluripotent stem cells.

PubMed

Reimer, Andreas; Vasilevich, Aliaksei; Hulshof, Frits; Viswanathan, Priyalakshmi; van Blitterswijk, Clemens A; de Boer, Jan; Watt, Fiona M

2016-01-13

It is well established that topographical features modulate cell behaviour, including cell morphology, proliferation and differentiation. To define the effects of topography on human induced pluripotent stem cells (iPSC), we plated cells on a topographical library containing over 1000 different features in medium lacking animal products (xeno-free). Using high content imaging, we determined the effect of each topography on cell proliferation and expression of the pluripotency marker Oct4 24 h after seeding. Features that maintained Oct4 expression also supported proliferation and cell-cell adhesion at 24 h, and by 4 days colonies of Oct4-positive, Sox2-positive cells had formed. Computational analysis revealed that small feature size was the most important determinant of pluripotency, followed by high wave number and high feature density. Using this information we correctly predicted whether any given topography within our library would support the pluripotent state at 24 h. This approach not only facilitates the design of substrates for optimal human iPSC expansion, but also, potentially, identification of topographies with other desirable characteristics, such as promoting differentiation.

Effects of genetic polymorphisms on the OCT1 and OCT2-mediated uptake of ranitidine.

PubMed

Meyer, Marleen Julia; Seitz, Tina; Brockmöller, Jürgen; Tzvetkov, Mladen Vassilev

2017-01-01

Ranitidine (Zantac®) is a H2-receptor antagonist commonly used for the treatment of acid-related gastrointestinal diseases. Ranitidine was reported to be a substrate of the organic cation transporters OCT1 and OCT2. The hepatic transporter OCT1 is highly genetically variable. Twelve major alleles confer partial or complete loss of OCT1 activity. The effects of these polymorphisms are highly substrate-specific and therefore difficult to predict. The renal transporter OCT2 has a common polymorphism, Ala270Ser, which was reported to affect OCT2 activity. In this study we analyzed the effects of genetic polymorphisms in OCT1 and OCT2 on the uptake of ranitidine and on its potency to inhibit uptake of other drugs. We characterized ranitidine uptake using HEK293 and CHO cells stably transfected to overexpress wild type OCT1, OCT2, or their naturally occurring allelic variants. Ranitidine was transported by wild-type OCT1 with a Km of 62.9 μM and a vmax of 1125 pmol/min/mg protein. Alleles OCT1*5, *6, *12, and *13 completely lacked ranitidine uptake. Alleles OCT1*2, *3, *4, and *10 had vmax values decreased by more than 50%. In contrast, OCT1*8 showed an increase of vmax by 25%. The effects of OCT1 alleles on ranitidine uptake strongly correlated with the effects on morphine uptake suggesting common interaction mechanisms of both drugs with OCT1. Ranitidine inhibited the OCT1-mediated uptake of metformin and morphine at clinically relevant concentrations. The inhibitory potency for morphine uptake was affected by the OCT1*2 allele. OCT2 showed only a limited uptake of ranitidine that was not significantly affected by the Ala270Ser polymorphism. We confirmed ranitidine as an OCT1 substrate and demonstrated that common genetic polymorphisms in OCT1 strongly affect ranitidine uptake and modulate ranitidine's potential to cause drug-drug interactions. The effects of the frequent OCT1 polymorphisms on ranitidine pharmacokinetics in humans remain to be analyzed.

Diclofenac pretreatment modulates exercise-induced inflammation in skeletal muscle of rats through the TLR4/NF-κB pathway.

PubMed

Barcelos, Rômulo Pillon; Bresciani, Guilherme; Cuevas, Maria José; Martínez-Flórez, Susana; Soares, Félix Alexandre Antunes; González-Gallego, Javier

2017-07-01

Nonsteroidal anti-inflammatory drugs, such as diclofenac, are widely used to treat inflammation and pain in several conditions, including sports injuries. This study analyzes the influence of diclofenac on the toll-like receptor-nuclear factor kappa B (TLR-NF-κB) pathway in skeletal muscle of rats submitted to acute eccentric exercise. Twenty male Wistar rats were divided into 4 groups: control-saline, control-diclofenac, exercise-saline, and exercise-diclofenac. Diclofenac or saline were administered for 7 days prior to an acute eccentric exercise bout. The inflammatory status was evaluated through mRNA levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), IL-1β, and tumor necrosis factor alpha (TNF-α), and protein content of COX-2, IL-6, and TNF-α in vastus lateralis muscle. Data obtained showed that a single bout of eccentric exercise significantly increased COX-2 gene expression. Similarly, mRNA expression and protein content of other inflammation-related genes also increased after the acute exercise. However, these effects were attenuated in the exercise + diclofenac group. TLR4, myeloid differentiation primary response gene 88 (MyD88), and p65 were also upregulated after the acute eccentric bout and the effect was blunted by the anti-inflammatory drug. These findings suggest that pretreatment with diclofenac may represent an effective tool to ameliorate the pro-inflammatory status induced by acute exercise in rat skeletal muscle possibly through an attenuation of the TLR4-NF-κB signaling pathway.

Genetic variants of organic cation transporter 1 (OCT1) and OCT2 significantly reduce lamivudine uptake.

PubMed

Choi, Min-Koo; Song, Im-Sook

2012-04-01

The study sought to investigate the effect of genetic variants of OCT1 (OCT1-P283L and -P341L) and OCT2 (OCT2-T199I, -T201M and -A270S), which were identified in a Korean population, on the transport of lamivudine in vitro and to compare the substrate dependent effects of OCT1 and OCT2 variants with 1-methyl-4-phenylpyridinium (MPP+), tetraethyl ammonium (TEA), metformin and lamivudine as substrates for these transporters. When the transport kinetics of lamivudine uptake in oocytes overexpressing OCT1 and OCT2 wild-type (WT) and variant proteins were measured, lamivudine uptake mediated by OCT1-WT was saturable, and uptake was decreased in oocytes expressing OCT1-P283L and -P341L variants compared with that in OCT1-WT. The Clint of lamivudine in oocytes expressing OCT1-P283L was decreased by 85.1% compared with OCT1-WT, whereas it was decreased by 48.7% in oocytes expressing OCT1-P341L. The Clint of lamivudine in oocytes expressing OCT2-T199I, -T201M and -A270S was decreased by 86.2%, 88.9% and 73.6%, respectively, compared with OCT2-WT. When comparing various substrates such as MPP+, TEA, metformin and lamivudine, the effects of the OCT1 genetic polymorphisms on their uptake were not identical. However, contrary to the case of OCT1, the uptake of MPP+, TEA, metformin and lamivudine in oocytes expressing OCT2-T199I, -T201M and -A270S variants was decreased significantly compared with that in oocytes expressing OCT2-WT. In conclusion, the effect of genetic variations of OCT1 and OCT2 on the uptake of MPP+, TEA, metformin and lamivudine was substrate-dependent. Copyright © 2012 John Wiley & Sons, Ltd.

Wide-Field Megahertz OCT Imaging of Patients with Diabetic Retinopathy.

PubMed

Reznicek, Lukas; Kolb, Jan P; Klein, Thomas; Mohler, Kathrin J; Wieser, Wolfgang; Huber, Robert; Kernt, Marcus; Märtz, Josef; Neubauer, Aljoscha S

2015-01-01

To evaluate the feasibility of wide-field Megahertz (MHz) OCT imaging in patients with diabetic retinopathy. A consecutive series of 15 eyes of 15 patients with diagnosed diabetic retinopathy were included. All patients underwent Megahertz OCT imaging, a close clinical examination, slit lamp biomicroscopy, and funduscopic evaluation. To acquire densely sampled, wide-field volumetric datasets, an ophthalmic 1050 nm OCT prototype system based on a Fourier-domain mode-locked (FDML) laser source with 1.68 MHz A-scan rate was employed. RESULTS. We were able to obtain OCT volume scans from all included 15 patients. Acquisition time was 1.8 seconds. Obtained volume datasets consisted of 2088 × 1044 A-scans of 60° of view. Thus, reconstructed en face images had a resolution of 34.8 pixels per degree in x-axis and 17.4 pixels per degree. Due to the densely sampled OCT volume dataset, postprocessed customized cross-sectional B-frames through pathologic changes such as an individual microaneurysm or a retinal neovascularization could be imaged. Wide-field Megahertz OCT is feasible to successfully image patients with diabetic retinopathy at high scanning rates and a wide angle of view, providing information in all three axes. The Megahertz OCT is a useful tool to screen diabetic patients for diabetic retinopathy.

Regulation of Kruppel-like factor 4, 9, and 13 genes and the steroidogenic genes LDLR, StAR, and CYP11A in ovarian granulosa cells.

PubMed

Natesampillai, Sekar; Kerkvliet, Jason; Leung, Peter C K; Veldhuis, Johannes D

2008-02-01

Kruppel-like factors (KLFs) are important Sp1-like eukaryotic transcriptional proteins. The LDLR, StAR, and CYP11A genes exhibit GC-rich Sp1-like sites, which have the potential to bind KLFs in multiprotein complexes. We now report that KLF4, KLF9, and KLF13 transcripts are expressed in and regulate ovarian cells. KLF4 and 13, but not KLF9, mRNA expression was induced and then repressed over time (P < 0.001). Combined LH and IGF-I stimulation increased KLF4 mRNA at 2 h (P < 0.01), whereas LH decreased KLF13 mRNA at 6 h (P < 0.05), and IGF-I reduced KLF13 at 24 h (P < 0.01) compared with untreated control. KLF9 was not regulated by either hormone. Transient transfection of KLF4, KLF9, and KLF13 suppressed LDLR/luc, StAR/luc, and CYP11A/luc by 80-90% (P < 0.001). Histone-deacetylase (HDAC) inhibitors stimulated LDLR/luc five- to sixfold and StAR/luc and CYP11A/luc activity twofold (P < 0.001) and partially reversed suppression by all three KLFs (P < 0.001). Deletion of the zinc finger domain of KLF13 abrogated repression of LDLR/luc. Lentiviral overexpression of the KLF13 gene suppressed LDLR mRNA (P < 0.001) and CYP11A mRNA (P = 0.003) but increased StAR mRNA (P = 0.007). Collectively, these data suggest that KLFs may recruit inhibitory complexes containing HDAC corepressors, thereby repressing LDLR and CYP11A transcription. Conversely, KLF13 may recruit unknown coactivators or stabilize StAR mRNA, thereby explaining enhancement of in situ StAR gene expression. These data introduce new potent gonadal transregulators of genes encoding proteins that mediate sterol uptake and steroid biosynthesis.

[Expression of the Drosophila melanogaster limk1 gene 3'-UTRs mRNA in Yeast Saccharomyces cerevisiae].

PubMed

Rumyantsev, A M; Zakharov, G A; Zhuravlev, A V; Padkina, M V; Savvateeva-Popova, E V; Sambuk, E V

2014-06-01

The stability of mRNA and its translation efficacy in higher eukaryotes are influenced by the interaction of 3'-untranscribed regions (3'-UTRs) with microRNAs and RNA-binding proteins. Since Saccharomyces cerevisiae lack microRNAs, it is possible to evaluate the contribution of only 3'-UTRs' and RNA-binding proteins' interaction in post-transcriptional regulation. For this, the post-transcriptional regulation of Drosophila limk1 gene encoding for the key enzyme of actin remodeling was studied in yeast. Analysis of limkl mRNA 3'-UTRs revealed the potential sites of yeast transcriptional termination. Computer remodeling demonstrated the possibility of secondary structure formation in limkl mRNA 3'-UTRs. For an evaluation of the functional activity of Drosophila 3'-UTRs in yeast, the reporter gene PHO5 encoding for yeast acid phosphatase (AP) fused to different variants of Drosophila limk1 mRNA 3'-UTRs (513, 1075, 1554 bp) was used. Assessments of AP activity and RT-PCR demonstrated that Drosophila limkl gene 3'-UTRs were functionally active and recognized in yeast. Therefore, yeast might be used as an appropriate model system for studies of 3'-UTR's role in post-transcriptional regulation.

Coupled Evolution of Transcription and mRNA Degradation

PubMed Central

Dori-Bachash, Mally; Shema, Efrat; Tirosh, Itay

2011-01-01

mRNA levels are determined by the balance between transcription and mRNA degradation, and while transcription has been extensively studied, very little is known regarding the regulation of mRNA degradation and its coordination with transcription. Here we examine the evolution of mRNA degradation rates between two closely related yeast species. Surprisingly, we find that around half of the evolutionary changes in mRNA degradation were coupled to transcriptional changes that exert opposite effects on mRNA levels. Analysis of mRNA degradation rates in an interspecific hybrid further suggests that opposite evolutionary changes in transcription and in mRNA degradation are mechanistically coupled and were generated by the same individual mutations. Coupled changes are associated with divergence of two complexes that were previously implicated both in transcription and in mRNA degradation (Rpb4/7 and Ccr4-Not), as well as with sequence divergence of transcription factor binding motifs. These results suggest that an opposite coupling between the regulation of transcription and that of mRNA degradation has shaped the evolution of gene regulation in yeast. PMID:21811398

Nuclear Orphan Receptor TLX Induces Oct-3/4 for the Survival and Maintenance of Adult Hippocampal Progenitors upon Hypoxia*

PubMed Central

Chavali, Pavithra Lakshminarasimhan; Saini, Ravi Kanth Rao; Matsumoto, Yoshiki; Ågren, Hans; Funa, Keiko

2011-01-01

Hypoxia promotes neural stem cell proliferation, the mechanism of which is poorly understood. Here, we have identified the nuclear orphan receptor TLX as a mediator for proliferation and pluripotency of neural progenitors upon hypoxia. We found an enhanced early protein expression of TLX under hypoxia potentiating sustained proliferation of neural progenitors. Moreover, TLX induction upon hypoxia in differentiating conditions leads to proliferation and a stem cell-like phenotype, along with coexpression of neural stem cell markers. Following hypoxia, TLX is recruited to the Oct-3/4 proximal promoter, augmenting the gene transcription and promoting progenitor proliferation and pluripotency. Knockdown of Oct-3/4 significantly reduced TLX-mediated proliferation, highlighting their interdependence in regulating the progenitor pool. Additionally, TLX synergizes with basic FGF to sustain cell viability upon hypoxia, since the knockdown of TLX along with the withdrawal of growth factor results in cell death. This can be attributed to the activation of Akt signaling pathway by TLX, the depletion of which results in reduced proliferation of progenitor cells. Cumulatively, the data presented here demonstrate a new role for TLX in neural stem cell proliferation and pluripotency upon hypoxia. PMID:21135096

Nuclear orphan receptor TLX induces Oct-3/4 for the survival and maintenance of adult hippocampal progenitors upon hypoxia.

PubMed

Chavali, Pavithra Lakshminarasimhan; Saini, Ravi Kanth Rao; Matsumoto, Yoshiki; Ågren, Hans; Funa, Keiko

2011-03-18

Hypoxia promotes neural stem cell proliferation, the mechanism of which is poorly understood. Here, we have identified the nuclear orphan receptor TLX as a mediator for proliferation and pluripotency of neural progenitors upon hypoxia. We found an enhanced early protein expression of TLX under hypoxia potentiating sustained proliferation of neural progenitors. Moreover, TLX induction upon hypoxia in differentiating conditions leads to proliferation and a stem cell-like phenotype, along with coexpression of neural stem cell markers. Following hypoxia, TLX is recruited to the Oct-3/4 proximal promoter, augmenting the gene transcription and promoting progenitor proliferation and pluripotency. Knockdown of Oct-3/4 significantly reduced TLX-mediated proliferation, highlighting their interdependence in regulating the progenitor pool. Additionally, TLX synergizes with basic FGF to sustain cell viability upon hypoxia, since the knockdown of TLX along with the withdrawal of growth factor results in cell death. This can be attributed to the activation of Akt signaling pathway by TLX, the depletion of which results in reduced proliferation of progenitor cells. Cumulatively, the data presented here demonstrate a new role for TLX in neural stem cell proliferation and pluripotency upon hypoxia.

Photobiomodulation effects on mRNA levels from genomic and chromosome stabilization genes in injured muscle.

PubMed

da Silva Neto Trajano, Larissa Alexsandra; Trajano, Eduardo Tavares Lima; da Silva Sergio, Luiz Philippe; Teixeira, Adilson Fonseca; Mencalha, Andre Luiz; Stumbo, Ana Carolina; de Souza da Fonseca, Adenilson

2018-04-26

Muscle injuries are the most prevalent type of injury in sports. A great number of athletes have relapsed in muscle injuries not being treated properly. Photobiomodulation therapy is an inexpensive and safe technique with many benefits in muscle injury treatment. However, little has been explored about the infrared laser effects on DNA and telomeres in muscle injuries. Thus, the aim of this study was to evaluate photobiomodulation effects on mRNA relative levels from genes related to telomere and genomic stabilization in injured muscle. Wistar male rats were randomly divided into six groups: control, laser 25 mW, laser 75 mW, injury, injury laser 25 mW, and injury laser 75 mW. Photobiomodulation was performed with 904 nm, 3 J/cm 2 at 25 or 75 mW. Cryoinjury was induced by two applications of a metal probe cooled in liquid nitrogen directly on the tibialis anterior muscle. After euthanasia, skeletal muscle samples were withdrawn and total RNA extracted for evaluation of mRNA levels from genomic (ATM and p53) and chromosome stabilization (TRF1 and TRF2) genes by real-time quantitative polymerization chain reaction. Data show that photobiomodulation reduces the mRNA levels from ATM and p53, as well reduces mRNA levels from TRF1 and TRF2 at 25 and 75 mW in injured skeletal muscle. In conclusion, photobiomodulation alters mRNA relative levels from genes related to genomic and telomere stabilization in injured skeletal muscle.

Jolkinolide B induces apoptosis and inhibits tumor growth in mouse melanoma B16F10 cells by altering glycolysis.

PubMed

Gao, Caixia; Yan, Xinyan; Wang, Bo; Yu, Lina; Han, Jichun; Li, Defang; Zheng, Qiusheng

2016-10-31

Most cancer cells preferentially rely on glycolysis to produce the energy (adenosine triphosphate, ATP) for growth and proliferation. Emerging evidence demonstrates that the apoptosis in cancer cells could be closely associated with the inhibition of glycolysis. In this study, we have found that jolkinolide B (JB), a bioactive diterpenoid extracted from the root of Euphorbia fischeriana Steud, induced tumor cells apoptosis and decreased the production of ATP and lactic acid in mouse melanoma B16F10 cells. Furthermore, we found that JB downregulated the mRNA expression of glucose transporter genes (Glut1, Glut3 and Glut4) and glycolysis-related kinase genes (Hk2 and Ldha) in B16F10 cells. Moreover, treatment with JB upregulated the mRNA expression of pro-apoptosis genes (Bax), downregulated the mRNA expression of anti-apoptosis genes (Bcl-2, Caspase-3 and Caspase-9), decreased the potential of mitochondrial membrane and increased reactive oxygen species (ROS) levels in B16F10 cells. Finally, intragastric administration of JB suppressed tumor growth and induced tumor apoptosis in mouse xenograft model of murine melanoma B16F10 cells. Taken together, these results suggest that JB could induce apoptosis through the mitochondrial pathway and inhibit tumor growth. The inhibition of glycolysis could play a crucial role in the induction of apoptosis in JB-treated B16F10 cells.

Jolkinolide B induces apoptosis and inhibits tumor growth in mouse melanoma B16F10 cells by altering glycolysis

PubMed Central

Gao, Caixia; Yan, Xinyan; Wang, Bo; Yu, Lina; Han, Jichun; Li, Defang; Zheng, Qiusheng

2016-01-01

Most cancer cells preferentially rely on glycolysis to produce the energy (adenosine triphosphate, ATP) for growth and proliferation. Emerging evidence demonstrates that the apoptosis in cancer cells could be closely associated with the inhibition of glycolysis. In this study, we have found that jolkinolide B (JB), a bioactive diterpenoid extracted from the root of Euphorbia fischeriana Steud, induced tumor cells apoptosis and decreased the production of ATP and lactic acid in mouse melanoma B16F10 cells. Furthermore, we found that JB downregulated the mRNA expression of glucose transporter genes (Glut1, Glut3 and Glut4) and glycolysis-related kinase genes (Hk2 and Ldha) in B16F10 cells. Moreover, treatment with JB upregulated the mRNA expression of pro-apoptosis genes (Bax), downregulated the mRNA expression of anti-apoptosis genes (Bcl-2, Caspase-3 and Caspase-9), decreased the potential of mitochondrial membrane and increased reactive oxygen species (ROS) levels in B16F10 cells. Finally, intragastric administration of JB suppressed tumor growth and induced tumor apoptosis in mouse xenograft model of murine melanoma B16F10 cells. Taken together, these results suggest that JB could induce apoptosis through the mitochondrial pathway and inhibit tumor growth. The inhibition of glycolysis could play a crucial role in the induction of apoptosis in JB-treated B16F10 cells. PMID:27796318

Effects of genetic polymorphisms on the OCT1 and OCT2-mediated uptake of ranitidine

PubMed Central

Meyer, Marleen Julia; Seitz, Tina; Brockmöller, Jürgen

2017-01-01

Background Ranitidine (Zantac®) is a H2-receptor antagonist commonly used for the treatment of acid-related gastrointestinal diseases. Ranitidine was reported to be a substrate of the organic cation transporters OCT1 and OCT2. The hepatic transporter OCT1 is highly genetically variable. Twelve major alleles confer partial or complete loss of OCT1 activity. The effects of these polymorphisms are highly substrate-specific and therefore difficult to predict. The renal transporter OCT2 has a common polymorphism, Ala270Ser, which was reported to affect OCT2 activity. Aim In this study we analyzed the effects of genetic polymorphisms in OCT1 and OCT2 on the uptake of ranitidine and on its potency to inhibit uptake of other drugs. Methods and results We characterized ranitidine uptake using HEK293 and CHO cells stably transfected to overexpress wild type OCT1, OCT2, or their naturally occurring allelic variants. Ranitidine was transported by wild-type OCT1 with a Km of 62.9 μM and a vmax of 1125 pmol/min/mg protein. Alleles OCT1*5, *6, *12, and *13 completely lacked ranitidine uptake. Alleles OCT1*2, *3, *4, and *10 had vmax values decreased by more than 50%. In contrast, OCT1*8 showed an increase of vmax by 25%. The effects of OCT1 alleles on ranitidine uptake strongly correlated with the effects on morphine uptake suggesting common interaction mechanisms of both drugs with OCT1. Ranitidine inhibited the OCT1-mediated uptake of metformin and morphine at clinically relevant concentrations. The inhibitory potency for morphine uptake was affected by the OCT1*2 allele. OCT2 showed only a limited uptake of ranitidine that was not significantly affected by the Ala270Ser polymorphism. Conclusions We confirmed ranitidine as an OCT1 substrate and demonstrated that common genetic polymorphisms in OCT1 strongly affect ranitidine uptake and modulate ranitidine’s potential to cause drug-drug interactions. The effects of the frequent OCT1 polymorphisms on ranitidine

Group II intron inhibits conjugative relaxase expression in bacteria by mRNA targeting

PubMed Central

Piazza, Carol Lyn; Smith, Dorie

2018-01-01

Group II introns are mobile ribozymes that are rare in bacterial genomes, often cohabiting with various mobile elements, and seldom interrupting housekeeping genes. What accounts for this distribution has not been well understood. Here, we demonstrate that Ll.LtrB, the group II intron residing in a relaxase gene on a conjugative plasmid from Lactococcus lactis, inhibits its host gene expression and restrains the naturally cohabiting mobile element from conjugative horizontal transfer. We show that reduction in gene expression is mainly at the mRNA level, and results from the interaction between exon-binding sequences (EBSs) in the intron and intron-binding sequences (IBSs) in the mRNA. The spliced intron targets the relaxase mRNA and reopens ligated exons, causing major mRNA loss. Taken together, this study provides an explanation for the distribution and paucity of group II introns in bacteria, and suggests a potential force for those introns to evolve into spliceosomal introns. PMID:29905149

Group II intron inhibits conjugative relaxase expression in bacteria by mRNA targeting.

PubMed

Qu, Guosheng; Piazza, Carol Lyn; Smith, Dorie; Belfort, Marlene

2018-06-15

Group II introns are mobile ribozymes that are rare in bacterial genomes, often cohabiting with various mobile elements, and seldom interrupting housekeeping genes. What accounts for this distribution has not been well understood. Here, we demonstrate that Ll.LtrB, the group II intron residing in a relaxase gene on a conjugative plasmid from Lactococcus lactis , inhibits its host gene expression and restrains the naturally cohabiting mobile element from conjugative horizontal transfer. We show that reduction in gene expression is mainly at the mRNA level, and results from the interaction between exon-binding sequences (EBSs) in the intron and intron-binding sequences (IBSs) in the mRNA. The spliced intron targets the relaxase mRNA and reopens ligated exons, causing major mRNA loss. Taken together, this study provides an explanation for the distribution and paucity of group II introns in bacteria, and suggests a potential force for those introns to evolve into spliceosomal introns. © 2018, Qu et al.

Effects of ACTH, dexamethasone, and adrenalectomy on 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) gene expression in the rat central nervous system

PubMed Central

Ye, Ping; Kenyon, Christopher J; MacKenzie, Scott M; Nichol, Katherine; Seckl, Jonathan R; Fraser, Robert; Connell, John M C; Davies, Eleanor

2008-01-01

Using a highly sensitive quantitative RT-PCR method for the measurement of CYP11B1 (11β-hydroxylase) and CYP11B2 (aldosterone synthase) mRNAs, we previously demonstrated that CYP11B2 expression in the central nervous system (CNS) is subject to regulation by dietary sodium. We have now quantified the expression of these genes in the CNS of male Wistar Kyoto (WKY) rats in response to systemic ACTH infusion, dexamethasone infusion, and to adrenalectomy. CYP11B1 and CYP11B2 mRNA levels were measured in total RNA isolated from the adrenal gland and discrete brain regions using real-time quantitative RT-PCR. ACTH infusion (40 ng/day for 7 days, N=8) significantly increased CYP11B1 mRNA in the adrenal gland, hypothalamus, and cerebral cortex compared with animals infused with vehicle only. ACTH infusion decreased adrenal CYP11B2 expression but increased expression in all of the CNS regions except the cortex. Dexamethasone (10 μg/day for 7 days, N=8) reduced adrenal CYP11B1 mRNA compared with control animals but had no significant effect on either gene's expression in the CNS. Adrenalectomy (N=6 per group) significantly increased CYP11B1 expression in the hippocampus and hypothalamus and raised CYP11B2 expression in the cerebellum relative to sham-operated animals. This study confirms the transcription of CYP11B1 and CYP11B2 throughout the CNS and demonstrates that gene transcription is subject to differential regulation by ACTH and circulating corticosteroid levels. PMID:18252953

Integrating mRNA and miRNA Weighted Gene Co-Expression Networks with eQTLs in the Nucleus Accumbens of Subjects with Alcohol Dependence

PubMed Central

Blevins, Tana; Aliev, Fazil; Adkins, Amy; Hack, Laura; Bigdeli, Tim; D. van der Vaart, Andrew; Web, Bradley Todd; Bacanu, Silviu-Alin; Kalsi, Gursharan; Kendler, Kenneth S.; Miles, Michael F.; Dick, Danielle; Riley, Brien P.; Dumur, Catherine; Vladimirov, Vladimir I.

2015-01-01

Alcohol consumption is known to lead to gene expression changes in the brain. After performing weighted gene co-expression network analyses (WGCNA) on genome-wide mRNA and microRNA (miRNA) expression in Nucleus Accumbens (NAc) of subjects with alcohol dependence (AD; N = 18) and of matched controls (N = 18), six mRNA and three miRNA modules significantly correlated with AD were identified (Bonferoni-adj. p≤ 0.05). Cell-type-specific transcriptome analyses revealed two of the mRNA modules to be enriched for neuronal specific marker genes and downregulated in AD, whereas the remaining four mRNA modules were enriched for astrocyte and microglial specific marker genes and upregulated in AD. Gene set enrichment analysis demonstrated that neuronal specific modules were enriched for genes involved in oxidative phosphorylation, mitochondrial dysfunction and MAPK signaling. Glial-specific modules were predominantly enriched for genes involved in processes related to immune functions, i.e. cytokine signaling (all adj. p≤ 0.05). In mRNA and miRNA modules, 461 and 25 candidate hub genes were identified, respectively. In contrast to the expected biological functions of miRNAs, correlation analyses between mRNA and miRNA hub genes revealed a higher number of positive than negative correlations (χ2 test p≤ 0.0001). Integration of hub gene expression with genome-wide genotypic data resulted in 591 mRNA cis-eQTLs and 62 miRNA cis-eQTLs. mRNA cis-eQTLs were significantly enriched for AD diagnosis and AD symptom counts (adj. p = 0.014 and p = 0.024, respectively) in AD GWAS signals in a large, independent genetic sample from the Collaborative Study on Genetics of Alcohol (COGA). In conclusion, our study identified putative gene network hubs coordinating mRNA and miRNA co-expression changes in the NAc of AD subjects, and our genetic (cis-eQTL) analysis provides novel insights into the etiological mechanisms of AD. PMID:26381263

Scalable topographies to support proliferation and Oct4 expression by human induced pluripotent stem cells

PubMed Central

Reimer, Andreas; Vasilevich, Aliaksei; Hulshof, Frits; Viswanathan, Priyalakshmi; van Blitterswijk, Clemens A.; de Boer, Jan; Watt, Fiona M.

2016-01-01

It is well established that topographical features modulate cell behaviour, including cell morphology, proliferation and differentiation. To define the effects of topography on human induced pluripotent stem cells (iPSC), we plated cells on a topographical library containing over 1000 different features in medium lacking animal products (xeno-free). Using high content imaging, we determined the effect of each topography on cell proliferation and expression of the pluripotency marker Oct4 24 h after seeding. Features that maintained Oct4 expression also supported proliferation and cell-cell adhesion at 24 h, and by 4 days colonies of Oct4-positive, Sox2-positive cells had formed. Computational analysis revealed that small feature size was the most important determinant of pluripotency, followed by high wave number and high feature density. Using this information we correctly predicted whether any given topography within our library would support the pluripotent state at 24 h. This approach not only facilitates the design of substrates for optimal human iPSC expansion, but also, potentially, identification of topographies with other desirable characteristics, such as promoting differentiation. PMID:26757610

The mRNA cap-binding protein Cbc1 is required for high and timely expression of genes by promoting the accumulation of gene-specific activators at promoters.

PubMed

Li, Tianlu; De Clercq, Nikki; Medina, Daniel A; Garre, Elena; Sunnerhagen, Per; Pérez-Ortín, José E; Alepuz, Paula

2016-02-01

The highly conserved Saccharomyces cerevisiae cap-binding protein Cbc1/Sto1 binds mRNA co-transcriptionally and acts as a key coordinator of mRNA fate. Recently, Cbc1 has also been implicated in transcription elongation and pre-initiation complex (PIC) formation. Previously, we described Cbc1 to be required for cell growth under osmotic stress and to mediate osmostress-induced translation reprogramming. Here, we observe delayed global transcription kinetics in cbc1Δ during osmotic stress that correlates with delayed recruitment of TBP and RNA polymerase II to osmo-induced promoters. Interestingly, we detect an interaction between Cbc1 and the MAPK Hog1, which controls most gene expression changes during osmostress, and observe that deletion of CBC1 delays the accumulation of the activator complex Hot1-Hog1 at osmostress promoters. Additionally, CBC1 deletion specifically reduces transcription rates of highly transcribed genes under non-stress conditions, such as ribosomal protein (RP) genes, while having low impact on transcription of weakly expressed genes. For RP genes, we show that recruitment of the specific activator Rap1, and subsequently TBP, to promoters is Cbc1-dependent. Altogether, our results indicate that binding of Cbc1 to the capped mRNAs is necessary for the accumulation of specific activators as well as PIC components at the promoters of genes whose expression requires high and rapid transcription. Copyright © 2016 Elsevier B.V. All rights reserved.

Structure of the gene encoding VGF, a nervous system-specific mRNA that is rapidly and selectively induced by nerve growth factor in PC12 cells.

PubMed

Salton, S R; Fischberg, D J; Dong, K W

1991-05-01

Nerve growth factor (NGF) plays a critical role in the development and survival of neurons in the peripheral nervous system. Following treatment with NGF but not epidermal growth factor, rat pheochromocytoma (PC12) cells undergo neural differentiation. We have cloned a nervous system-specific mRNA, NGF33.1, that is rapidly and relatively selectively induced by treatment of PC12 cells with NGF and basic fibroblast growth factor in comparison with epidermal growth factor. Analysis of the nucleic acid and predicted amino acid sequences of the NGF33.1 cDNA clone suggested that this clone corresponded to the NGF-inducible mRNA called VGF (A. Levi, J. D. Eldridge, and B. M. Paterson, Science 229:393-395, 1985; R. Possenti, J. D. Eldridge, B. M. Paterson, A. Grasso, and A. Levi, EMBO J. 8:2217-2223, 1989). We have used the NGF33.1 cDNA clone to isolate and characterize the VGF gene, and in this paper we report the complete sequence of the VGF gene, including 853 bases of 5' flank revealed TATAA and CCAAT elements, several GC boxes, and a consensus cyclic AMP response element-binding protein binding site. The VGF promoter contains sequences homologous to other NGF-inducible, neuronal promoters. We further show that VGF mRNA is induced in PC12 cells to a greater extent by depolarization and by phorbol-12-myristate-13-acetate treatment than by 8-bromo-cyclic AMP treatment. By Northern (RNA) and RNase protection analysis, VGF mRNA is detectable in embryonic and postnatal central and peripheral nervous tissues but not in a number of nonneural tissues. In the cascade of events which ultimately leads to the neural differentiation of NGF-treated PC12 cells, the VGF gene encodes the most rapidly and selectively regulated, nervous-system specific mRNA yet identified.

Differentiation of female Oct4-GFP embryonic stem cells into germ lineage cells.

PubMed

Ma, Xin; Li, Peng; Sun, Xiang; Sun, Yifeng; Hu, Rong; Yuan, Ping

2018-04-01

Due to high infertility ratio nowadays, it is essential to explore efficient ways of enhancing mammalian reproductivity, in particular female reproductivity. Using female Oct4-GFP embryonic stem cells, we mimic the in vivo development procedure to induce ES cells into epiblast cell-like cells (EpiLCs) and then primordial germ cell-like cells (PGCLCs). GFP positive PGCLCs that showed typical PGC markers and epigenetic modification were efficiently obtained. Further transplantation of the GFP positive PGCLC and native ovary cell mixture into ovary of infertile mice revealed that both MVH and GFP positive cells could be developed in ovary, but no later developmental stage germ cells were observed. This study suggested that Oct4-GFP ES cells may be only suitable for tracing early germ cell development. © 2018 International Federation for Cell Biology.

Quantification of silkworm coactivator of MBF1 mRNA by SYBR Green I real-time RT-PCR reveals tissue- and stage-specific transcription levels.

PubMed

Li, Guang-li; Roy, Bhaskar; Li, Xing-hua; Yue, Wan-fu; Wu, Xiao-feng; Liu, Jian-mei; Zhang, Chuan-xi; Miao, Yun-gen

2009-05-01

Transcriptional coactivators play a crucial role in gene transcription and expression. Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for transcriptional activation caused by DNA-binding activators, such as FTZ-F1 and GCN4. Until now, very few studies have been reported in the silkworm. We selected the Bombyx mori because it is a model insect and acts as an economic animal for silk industry. In this study, we conducted the quantitative analysis of MBF1 mRNA in silkworm B. mori L. with actin (A3) as internal standard by means of SYBR Green I real-time RT-PCR method. The total RNA was extracted from the silk gland, epidermis, fat body, and midguts of the fifth instar B. mori larvae. The mRNA was reverse transcripted, and the cDNA fragments of MBF1 mRNA and actin gene were amplified by RT-PCR using specific primers. MBF1 mRNA expression in different tissues of silkworm B. mori L. was quantified using standardized SYBR Green I RT-PCR. The results suggested MBF1 gene was expressed in all investigated organs but highly expressed in the silk gland, showing its relation to biosynthesis of silk proteins.

Effect of early addition of bone morphogenetic protein 5 (BMP5) to embryo culture medium on in vitro development and expression of developmentally important genes in bovine preimplantation embryos.

PubMed

García, Elina V; Miceli, Dora C; Rizo, Gabriela; Valdecantos, Pablo A; Barrera, Antonio D

2015-09-01

Previous studies have reported that bone morphogenetic protein 5 (BMP5) is differentially expressed in the isthmus of bovine oviducts and it is present in the oviductal fluid. However, the specific action of this factor is unknown. To evaluate whether BMP5 exerts some effect during early bovine embryo development, gene expression of BMP5, BMP receptors, and the effect of exogenous BMP5 on in vitro development and expression of developmentally important genes were assessed. In experiment 1, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from in vitro fertilization, were collected for analysis of BMP5 and BMP receptors (BMPR1A, BMPR1B, and BMPR2) messenger RNA (mRNA) expression. On the basis of previous results, in experiment 2, presumptive zygotes were cultured for the first 48 hours after insemination in CR1aa medium assaying three different treatments: (1) control (CR1aa); (2) vehicle control (CR1aa + 0.04 mM HCl), and (3) BMP5 treatment (CR1aa + 100 ng/mL of BMP5). The cleavage rate was evaluated 48 hours after insemination (Day 2), and then, embryos were transferred to CR1aa + 10% fetal bovine serum. The blastocyst rate was determined on Day 7. In experiment 3, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from control and BMP5-treated groups, were collected for analysis of ID2 (BMP target gene), OCT4, NANOG, and SOX2 (pluripotency genes) mRNA expression. BMP5 transcripts were not detectable in any of the embryonic stages examined, whereas the relative mRNA abundance of the three BMP receptors analyzed was greater in early embryo development stages before maternal-embryonic transition, raising the possibility of a direct effect of exogenous BMPs on the embryo during the first developmental period. Although early addition of 100 ng/mL of BMP5 to the embryo culture medium had no effect on the cleavage rate, a significantly higher proportion of cleaved embryos developed to the

Beyond the known functions of the CCR4-NOT complex in gene expression regulatory mechanisms: New structural insights to unravel CCR4-NOT mRNA processing machinery.

PubMed

Ukleja, Marta; Valpuesta, José María; Dziembowski, Andrzej; Cuellar, Jorge

2016-10-01

Large protein assemblies are usually the effectors of major cellular processes. The intricate cell homeostasis network is divided into numerous interconnected pathways, each controlled by a set of protein machines. One of these master regulators is the CCR4-NOT complex, which ultimately controls protein expression levels. This multisubunit complex assembles around a scaffold platform, which enables a wide variety of well-studied functions from mRNA synthesis to transcript decay, as well as other tasks still being identified. Solving the structure of the entire CCR4-NOT complex will help to define the distribution of its functions. The recently published three-dimensional reconstruction of the complex, in combination with the known crystal structures of some of the components, has begun to address this. Methodological improvements in structural biology, especially in cryoelectron microscopy, encourage further structural and protein-protein interaction studies, which will advance our comprehension of the gene expression machinery. © 2016 WILEY Periodicals, Inc.

JMJD3 suppresses stem cell-like characteristics in breast cancer cells by downregulation of Oct4 independently of its demethylase activity.

PubMed

Xun, Jing; Wang, Dekun; Shen, Long; Gong, Junbo; Gao, Ruifang; Du, Lingfang; Chang, Antao; Song, Xiangrong; Xiang, Rong; Tan, Xiaoyue

2017-03-28

Epigenetic regulator JMJD3 plays an important role in both tumor progression and somatic cell reprogramming. Here, we explored the effect of JMJD3 on the stem cell-like characteristics of breast cancer and its underlying mechanism involving stemness-related transcription factor Oct4. Our data revealed that, in breast cancer cells lines and an orthotopic xenograph mouse model of breast cancer, ectopic overexpression of JMJD3 suppressed stem cell-like characteristics of breast cancer cells, whereas knockdown of JMJD3 promoted these characteristics. Oct4 mediated the suppressive effects of JMJD3 on the stemness of breast cancer cells. The inhibitory effect of JMJD3 on Oct4 was independent of demethylase activity, but mediated via degradation of PHF20. Furthermore, we applied an agonist of the vitamin D receptor, paricalcitol, and found that it induced JMJD3 in breast cancer cells. Our data showed that administration of paricalcitol suppressed stem cell-like characteristics and Oct4 expression. Taken together, JMJD3 inhibits the stem cell-like characteristics in breast cancer by suppression of stemness factor Oct4 in a PHF20-dependent manner. Administration of paricalcitol leads to upregulation of JMJD3 that suppresses Oct4 expression and the stem cell-like characteristics in breast cancer.

Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage.

PubMed

Brok-Volchanskaya, Vera S; Kadyrov, Farid A; Sivogrivov, Dmitry E; Kolosov, Peter M; Sokolov, Andrey S; Shlyapnikov, Michael G; Kryukov, Valentine M; Granovsky, Igor E

2008-04-01

Homing endonucleases initiate nonreciprocal transfer of DNA segments containing their own genes and the flanking sequences by cleaving the recipient DNA. Bacteriophage T4 segB gene, which is located in a cluster of tRNA genes, encodes a protein of unknown function, homologous to homing endonucleases of the GIY-YIG family. We demonstrate that SegB protein is a site-specific endonuclease, which produces mostly 3' 2-nt protruding ends at its DNA cleavage site. Analysis of SegB cleavage sites suggests that SegB recognizes a 27-bp sequence. It contains 11-bp conserved sequence, which corresponds to a conserved motif of tRNA TpsiC stem-loop, whereas the remainder of the recognition site is rather degenerate. T4-related phages T2L, RB1 and RB3 contain tRNA gene regions that are homologous to that of phage T4 but lack segB gene and several tRNA genes. In co-infections of phages T4 and T2L, segB gene is inherited with nearly 100% of efficiency. The preferred inheritance depends absolutely on the segB gene integrity and is accompanied by the loss of the T2L tRNA gene region markers. We suggest that SegB is a homing endonuclease that functions to ensure spreading of its own gene and the surrounding tRNA genes among T4-related phages.

Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage

PubMed Central

Brok-Volchanskaya, Vera S.; Kadyrov, Farid A.; Sivogrivov, Dmitry E.; Kolosov, Peter M.; Sokolov, Andrey S.; Shlyapnikov, Michael G.; Kryukov, Valentine M.; Granovsky, Igor E.

2008-01-01

Homing endonucleases initiate nonreciprocal transfer of DNA segments containing their own genes and the flanking sequences by cleaving the recipient DNA. Bacteriophage T4 segB gene, which is located in a cluster of tRNA genes, encodes a protein of unknown function, homologous to homing endonucleases of the GIY-YIG family. We demonstrate that SegB protein is a site-specific endonuclease, which produces mostly 3′ 2-nt protruding ends at its DNA cleavage site. Analysis of SegB cleavage sites suggests that SegB recognizes a 27-bp sequence. It contains 11-bp conserved sequence, which corresponds to a conserved motif of tRNA TψC stem-loop, whereas the remainder of the recognition site is rather degenerate. T4-related phages T2L, RB1 and RB3 contain tRNA gene regions that are homologous to that of phage T4 but lack segB gene and several tRNA genes. In co-infections of phages T4 and T2L, segB gene is inherited with nearly 100% of efficiency. The preferred inheritance depends absolutely on the segB gene integrity and is accompanied by the loss of the T2L tRNA gene region markers. We suggest that SegB is a homing endonuclease that functions to ensure spreading of its own gene and the surrounding tRNA genes among T4-related phages. PMID:18281701

Differential accumulation of nif structural gene mRNA in Azotobacter vinelandii.

PubMed

Hamilton, Trinity L; Jacobson, Marty; Ludwig, Marcus; Boyd, Eric S; Bryant, Donald A; Dean, Dennis R; Peters, John W

2011-09-01

Northern analysis was employed to investigate mRNA produced by mutant strains of Azotobacter vinelandii with defined deletions in the nif structural genes and in the intergenic noncoding regions. The results indicate that intergenic RNA secondary structures effect the differential accumulation of transcripts, supporting the high Fe protein-to-MoFe protein ratio required for optimal diazotrophic growth.

Operational Transition from the First-Generation Heidelberg Spectralis Optical Coherence Tomography [OCT("OCT1")] to the Second-Generation Heidelberg Spectralis OCT("OCT2")

NASA Technical Reports Server (NTRS)

Mason, S.; Brunstetter, T.; Young, M.; Van Baalen, M.; Tarver, W.; Derrick, R.; Wells, J.; Dey, B.; Todd, K.; Smith, B.;

Arsenic Induces Polyadenylation of Canonical Histone mRNA by Down-regulating Stem-Loop-binding Protein Gene Expression*

PubMed Central

Brocato, Jason; Fang, Lei; Chervona, Yana; Chen, Danqi; Kiok, Kathrin; Sun, Hong; Tseng, Hsiang-Chi; Xu, Dazhong; Shamy, Magdy; Jin, Chunyuan; Costa, Max

2014-01-01

The replication-dependent histone genes are the only metazoan genes whose messenger RNA (mRNA) does not terminate with a poly(A) tail at the 3′-end. Instead, the histone mRNAs display a stem-loop structure at their 3′-end. Stem-loop-binding protein (SLBP) binds the stem-loop and regulates canonical histone mRNA metabolism. Here we report that exposure to arsenic, a carcinogenic metal, decreased cellular levels of SLBP by inducing its proteasomal degradation and inhibiting SLBP transcription via epigenetic mechanisms. Notably, arsenic exposure dramatically increased polyadenylation of canonical histone H3.1 mRNA possibly through down-regulation of SLBP expression. The polyadenylated H3.1 mRNA induced by arsenic was not susceptible to normal degradation that occurs at the end of S phase, resulting in continued presence into mitosis, increased total H3.1 mRNA, and increased H3 protein levels. Excess expression of canonical histones have been shown to increase sensitivity to DNA damage as well as increase the frequency of missing chromosomes and induce genomic instability. Thus, polyadenylation of canonical histone mRNA following arsenic exposure may contribute to arsenic-induced carcinogenesis. PMID:25266719

HuR (Elavl1) and HuB (Elavl2) Stabilize Matrix Metalloproteinase-9 mRNA During Seizure-Induced Mmp-9 Expression in Neurons

PubMed Central

Zybura-Broda, Katarzyna; Wolder-Gontarek, Malgorzata; Ambrozek-Latecka, Magdalena; Choros, Artur; Bogusz, Agnieszka; Wilemska-Dziaduszycka, Joanna; Rylski, Marcin

2018-01-01

Matrix metalloproteinase-9 (Mmp-9) is involved in different general and cell-type–specific processes, both in neuronal and non-neuronal cells. Moreover, it is implicated in an induction or progression of various human disorders, including diseases of the central nervous system. Mechanisms regulating activity-driven Mmp-9 expression in neurons are still not fully understood. Here, we show that stabilization of Mmp-9 mRNA is one of the factors responsible for the neuronal activity-evoked upregulation of Mmp-9 mRNA expression in hippocampal neurons. Furthermore, we demonstrate that the molecular mechanism related to this stabilization is dependent on the neuronal seizure-triggered transiently increased binding of the mRNA stability-inducing protein, HuR, to ARE1 and ARE4 motifs of the 3′UTR for Mmp-9 mRNA as well as the stably augmented association of another mRNA-stabilizing protein, HuB, to the ARE1 element of the 3′UTR. Intriguingly, we demonstrate further that both HuR and HuB are crucial for an incidence of Mmp-9 mRNA stabilization after neuronal activation. This study identifies Mmp-9 mRNA as the first HuB target regulated by mRNA stabilization in neurons. Moreover, these results are the first to describe an existence of HuR-dependent mRNA stabilization in neurons of the brain. PMID:29686606

Wide-Field Megahertz OCT Imaging of Patients with Diabetic Retinopathy

PubMed Central

Reznicek, Lukas; Kolb, Jan P.; Klein, Thomas; Mohler, Kathrin J.; Huber, Robert; Kernt, Marcus; Märtz, Josef; Neubauer, Aljoscha S.

2015-01-01

Purpose. To evaluate the feasibility of wide-field Megahertz (MHz) OCT imaging in patients with diabetic retinopathy. Methods. A consecutive series of 15 eyes of 15 patients with diagnosed diabetic retinopathy were included. All patients underwent Megahertz OCT imaging, a close clinical examination, slit lamp biomicroscopy, and funduscopic evaluation. To acquire densely sampled, wide-field volumetric datasets, an ophthalmic 1050 nm OCT prototype system based on a Fourier-domain mode-locked (FDML) laser source with 1.68 MHz A-scan rate was employed. Results. We were able to obtain OCT volume scans from all included 15 patients. Acquisition time was 1.8 seconds. Obtained volume datasets consisted of 2088 × 1044 A-scans of 60° of view. Thus, reconstructed en face images had a resolution of 34.8 pixels per degree in x-axis and 17.4 pixels per degree. Due to the densely sampled OCT volume dataset, postprocessed customized cross-sectional B-frames through pathologic changes such as an individual microaneurysm or a retinal neovascularization could be imaged. Conclusions. Wide-field Megahertz OCT is feasible to successfully image patients with diabetic retinopathy at high scanning rates and a wide angle of view, providing information in all three axes. The Megahertz OCT is a useful tool to screen diabetic patients for diabetic retinopathy. PMID:26273665

Methylation of CpG island of p14(ARK), p15(INK4b) and p16(INK4a) genes in coke oven workers.

PubMed

Zhang, H; Li, X; Ge, L; Yang, J; Sun, J; Niu, Q

2015-02-01

To detect the blood genomic DNA methylation in coke oven workers and find a possible early screening index for occupational lung cancer, 74 coke oven workers as the exposed group and 47 water pump workers as the controls were surveyed, and urine samples and peripheral blood mononuclear cells (PBMCs) were collected. Airborne benzo[a]pyrene (B[a]P) levels in workplace and urinary 1-hydroxypyrene (1-OH-Py) levels were determined by high-performance liquid chromatography. DNA damage of PBMCs and the p14(ARK), p15(INK4b) and p16(INK4a) gene CpG island methylation in the promoter region were detected by comet assay and methylation-specific polymerase chain reaction techniques, respectively. Results show that compared with the controls, concentration of airborne B[a]Ps was elevated in the coke plant, and urinary 1-OH-Py's level and DNA olive tail moment in comet assay were significantly increased in the coke oven workers, and p14(ARK), p15(INK4b) and p16(INK4a) gene methylation rates were also significantly increased. With the working years and urinary 1-OH-Py's level, the rates of p14(ARK) and p16(INK4a) gene methylation were significantly increased while that of p15(INK4b) gene methylation displayed no statistical change. We conclude that PBMCs' p14(ARK) and p16(INK4a) gene methylation may be used for screening and warning lung cancer in coke oven workers. © The Author(s) 2015.

Ethanol-induced changes in poly (ADP ribose) polymerase and neuronal developmental gene expression.

PubMed

Gavin, David P; Kusumo, Handojo; Sharma, Rajiv P; Guizzetti, Marina

2016-11-01

Prenatal alcohol exposure has profound effects on neuronal growth and development. Poly-ADP Ribose Polymerase (PARP) enzymes are perhaps unique in the field of epigenetics in that they directly participate in histone modifications, transcription factor modifications, DNA methylation/demethylation and are highly inducible by ethanol. It was our hypothesis that ethanol would induce PARP enzymatic activity leading to alterations in neurodevelopmental gene expression. Mouse E18 cortical neurons were treated with ethanol, PARP inhibitors, and nuclear hormone receptor transcription factor PPARγ agonists and antagonists. Subsequently, we measured PARP activity and changes in Bdnf, OKSM (Oct4, Klf4, Sox2, c-Myc), DNA methylating/demethylating factors, and Pparγ mRNA expression, promoter 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC), and PPARγ promoter binding. We found that ethanol reduced Bdnf4, 9a, and Klf4 mRNA expression, and increased c-Myc expression. These changes were reversed with a PARP inhibitor. In agreement with its role in DNA demethylation PARP inhibition increased 5MC levels at the c-Myc promoter. In addition, we found that inhibition of PARP enzymatic activity increased PPARγ promoter binding, and this corresponded to increased Bdnf and Klf4 mRNA expression. Our results suggest that PARP participates in DNA demethylation and reduces PPARγ promoter binding. The current study underscores the importance of PARP in ethanol-induced changes to neurodevelopmental gene expression. Published by Elsevier Ltd.

[Expression of SLP-2 mRNA in endometrial cancer and its significance].

PubMed

Feng, Wang-qin; Cui, Zhu-mei; Feng, Feng-zhi; Qi, Xiu-juan; Ding, Fang; Li, Wen-dong; Liu, Zhi-hua

2005-08-01

To characterize the differential expression of SLP-2 in endometrial cancer, and to study the effect of human SLP-2 gene on human endometrial cancer cell line. The expression of SLP-2 gene in 32 cases of endometrial cancer and 28 cases of normal endometrial tissues was examined by semi-quantitative RT-PCR. Eukaryotic expression vectors of sense and antisense SLP-2 were constructed and transfected into HEC-1B cell line using lipofectamine 2000 respectively. The morphological changes of the cell were observed by phase contrast microscopy. The cell growth was detected by methyl thiazolyl tetrazolium (MTT) assay, and the cell cycles were analyzed by flow cytometry. The expression of SLP-2 mRNA in endometrial cancer tissues was higher than that in normal endometrial tissues (1.6 +/- 0.7 vs 0.7 +/- 0.3, P < 0.05). Sense and antisense human SLP-2 constructs were transfected into HEC-1B cell line respectively. After being transfected with sense SLP-2, the expression of SLP-2 mRNA in HEC-1B cell line was increased by about 2.4 times that of the control group, the cell growth was accelerated, and the number of cells in G(1) phase was decreased by 12.5%, S phase was increased by 8.0%. After being transfected with antisense SLP-2, the expression of SLP-2 mRNA was declined 50%. The transfected cells showed slower growth, and the number of cells in G(1) phase was significantly increased by 10.5%, and S phase was declined by 9.8%. SLP-2 mRNA shows up-regulation in endometrial cancer tissues, and it may have some relationship with carcinogenesis of endometrial cancer.

Sperm 1: a POU-domain gene transiently expressed immediately before meiosis I in the male germ cell.

PubMed Central

Andersen, B; Pearse, R V; Schlegel, P N; Cichon, Z; Schonemann, M D; Bardin, C W; Rosenfeld, M G

1993-01-01

Members of the POU-domain gene family encode for transcriptional regulatory molecules that are important for terminal differentiation of several organ systems, including anterior pituitary, sensory neurons, and B lymphocytes. We have identified a POU-domain factor, referred to as sperm 1 (Sprm-1). This factor is most related to the transactivator Oct-3/4, which is expressed in the early embryo, primordial germ cells, and the egg. However, in contrast with Oct-3/4, rat Sprm-1 is selectively expressed during a 36- to 48-hr period immediately preceding meiosis I in male germ cells. Although the POU-domain of Sprm-1 is divergent from the POU-domains of Oct-1 and Oct-2, random-site-selection assay reveals that Sprm-1 preferentially binds to a specific variant of the classic octamer DNA-response element in which the optimal sequence differs from that preferred by Oct-1 and Pit-1. These data suggest that the Sprm-1 gene encodes a DNA-binding protein that may exert a regulatory function in meiotic events that are required for terminal differentiation of the male germ cell. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7902581

Feature tracking for automated volume of interest stabilization on 4D-OCT images

NASA Astrophysics Data System (ADS)

Laves, Max-Heinrich; Schoob, Andreas; Kahrs, Lüder A.; Pfeiffer, Tom; Huber, Robert; Ortmaier, Tobias

2017-03-01

A common representation of volumetric medical image data is the triplanar view (TV), in which the surgeon manually selects slices showing the anatomical structure of interest. In addition to common medical imaging such as MRI or computed tomography, recent advances in the field of optical coherence tomography (OCT) have enabled live processing and volumetric rendering of four-dimensional images of the human body. Due to the region of interest undergoing motion, it is challenging for the surgeon to simultaneously keep track of an object by continuously adjusting the TV to desired slices. To select these slices in subsequent frames automatically, it is necessary to track movements of the volume of interest (VOI). This has not been addressed with respect to 4DOCT images yet. Therefore, this paper evaluates motion tracking by applying state-of-the-art tracking schemes on maximum intensity projections (MIP) of 4D-OCT images. Estimated VOI location is used to conveniently show corresponding slices and to improve the MIPs by calculating thin-slab MIPs. Tracking performances are evaluated on an in-vivo sequence of human skin, captured at 26 volumes per second. Among investigated tracking schemes, our recently presented tracking scheme for soft tissue motion provides highest accuracy with an error of under 2.2 voxels for the first 80 volumes. Object tracking on 4D-OCT images enables its use for sub-epithelial tracking of microvessels for image-guidance.

A splice junction-targeted CRISPR approach (spJCRISPR) reveals human FOXO3B to be a protein-coding gene.

PubMed

Santo, Evan E; Paik, Jihye

2018-06-17

The rapid development of CRISPR technology is revolutionizing molecular approaches to the dissection of complex biological phenomena. Here we describe an alternative generally applicable implementation of the CRISPR-Cas9 system that allows for selective knockdown of extremely homologous genes. This strategy employs the lentiviral delivery of paired sgRNAs and nickase Cas9 (Cas9D10A) to achieve targeted deletion of splice junctions. This general strategy offers several advantages over standard single-guide exon-targeting CRISPR-Cas9 such as greatly reduced off-target effects, more restricted genomic editing, routine disruption of target gene mRNA expression and the ability to differentiate between closely related genes. Here we demonstrate the utility of this strategy by achieving selective knockdown of the highly homologous human genes FOXO3A and suspected pseudogene FOXO3B. We find the spJCRISPR strategy to efficiently and selectively disrupt FOXO3A and FOXO3B mRNA and protein expression; thus revealing that the human FOXO3B locus encodes a bona fide human gene. Unlike FOXO3A, we find the FOXO3B protein to be cytosolically localized in both the presence and absence of active Akt. The ability to selectively target and efficiently disrupt the expression of the closely-related FOXO3A and FOXO3B genes demonstrates the efficacy of the spJCRISPR approach. Copyright © 2018. Published by Elsevier B.V.

OCT imaging of craniofacial anatomy in xenopus embryos (Conference Presentation)

NASA Astrophysics Data System (ADS)

Deniz, Engin; Jonas, Stephan M.; Griffin, John; Hooper, Michael C.; Choma, Michael A.; Khokha, Mustafa K.

2016-03-01

The etiology of craniofacial defects is incompletely understood. The ability to obtain large amounts of gene sequence data from families affected by craniofacial defects is opening up new ways to understand molecular genetic etiological factors. One important link between gene sequence data and clinical relevance is biological research into candidate genes and molecular pathways. We present our recent research using OCT as a nondestructive phenotyping modality of craniofacial morphology in Xenopus embryos, an important animal model for biological research in gene and pathway discovery. We define 2D and 3D scanning protocols for a standardized approach to craniofacial imaging in Xenopus embryos. We define standard views and planar reconstructions for visualizing normal anatomy and landmarks. We compare these views and reconstructions to traditional histopathology using alcian blue staining. In addition to being 3D, nondestructive, and having much faster throughout, OCT can identify craniofacial features that are lost during traditional histopathological preparation. We also identify quantitative morphometric parameters to define normative craniofacial anatomy. We also note that craniofacial and cardiac defects are not infrequently present in the same patient (e.g velocardiofacial syndrome). Given that OCT excels at certain aspects of cardiac imaging in Xenopus embryos, our work highlights the potential of using OCT and Xenopus to study molecular genetic factors that impact both cardiac and craniofacial development.

Functional characterization of the organic cation transporters (OCTs) in human airway pulmonary epithelial cells.

PubMed

Ingoglia, Filippo; Visigalli, Rossana; Rotoli, Bianca Maria; Barilli, Amelia; Riccardi, Benedetta; Puccini, Paola; Dall'Asta, Valeria

2015-07-01

Organic cation transporters (OCT1-3) mediate the transport of organic cations including inhaled drugs across the cell membrane, although their role in lung epithelium hasn't been well understood yet. We address here the expression and functional activity of OCT1-3 in human airway epithelial cells A549, Calu-3 and NCl-H441. Kinetic and inhibition analyses, employing [(3)H]1-methyl-4-phenylpyridinium (MPP+) as substrate, and the compounds quinidine, prostaglandine E2 (PGE2) and corticosterone as preferential inhibitors of OCT1, OCT2, and OCT3, respectively, have been performed. A549 cells present a robust MPP+ uptake mediated by one high-affinity component (Km~50μM) which is identifiable with OCT3. Corticosterone, indeed, completely inhibits MPP+ transport, while quinidine and PGE2 are inactive and SLC22A3/OCT3 silencing with siRNA markedly lowers MPP+ uptake. Conversely, Calu-3 exhibits both a high (Km<20μM) and a low affinity (Km>0.6mM) transport components, referable to OCT3 and OCT1, respectively, as demonstrated by the inhibition analysis performed at proper substrate concentrations and confirmed by the use of specific siRNA. These transporters are active also when cells are grown under air-liquid interface (ALI) conditions. Only a very modest saturable MPP+ uptake is measurable in NCl-H441 cells and the inhibitory effect of quinidine points to OCT1 as the subtype functionally involved in this model. Finally, the characterization of MPP+ transport in human bronchial BEAS-2B cells suggests that OCT1 and OCT3 are operative. These findings could help to identify in vitro models to be employed for studies concerning the specific involvement of each transporter in drug transportation. Copyright © 2015 Elsevier B.V. All rights reserved.

Replacement of the yeast TRP4 3' untranslated region by a hammerhead ribozyme results in a stable and efficiently exported mRNA that lacks a poly(A) tail.

PubMed Central

Düvel, Katrin; Valerius, Oliver; Mangus, David A; Jacobson, Allan; Braus, Gerhard H

2002-01-01

The mRNA poly(A) tail serves different purposes, including the facilitation of nuclear export, mRNA stabilization, efficient translation, and, finally, specific degradation. The posttranscriptional addition of a poly(A) tail depends on sequence motifs in the 3' untranslated region (3' UTR) of the mRNA and a complex trans-acting protein machinery. In this study, we have replaced the 3' UTR of the yeast TRP4 gene with sequences encoding a hammerhead ribozyme that efficiently cleaves itself in vivo. Expression of the TRP4-ribozyme allele resulted in the accumulation of a nonpolyadenylated mRNA. Cells expressing the TRP4-ribozyme mRNA showed a reduced growth rate due to a reduction in Trp4p enzyme activity. The reduction in enzyme activity was not caused by inefficient mRNA export from the nucleus or mRNA destabilization. Rather, analyses of mRNA association with polyribosomes indicate that translation of the ribozyme-containing mRNA is impaired. This translational defect allows sufficient synthesis of Trp4p to support growth of trp4 cells, but is, nevertheless, of such magnitude as to activate the general control network of amino acid biosynthesis. PMID:12003493

Splicing factor SR34b mutation reduces cadmium tolerance in Arabidopsis by regulating iron-regulated transporter 1 gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Wentao; Du, Bojing; Liu, Di

Highlights: • Arabidopsis splicing factor SR34b gene is cadmium-inducible. • SR34b T-DNA insertion mutant is sensitive to cadmium due to high cadmium uptake. • SR34b is a regulator of cadmium transporter IRT1 at the posttranscription level. • These results highlight the roles of splicing factors in cadmium tolerance of plant. - Abstract: Serine/arginine-rich (SR) proteins are important splicing factors. However, the biological functions of plant SR proteins remain unclear especially in abiotic stresses. Cadmium (Cd) is a non-essential element that negatively affects plant growth and development. In this study, we provided clear evidence for SR gene involved in Cd tolerancemore » in planta. Systemic expression analysis of 17 Arabidopsis SR genes revealed that SR34b is the only SR gene upregulated by Cd, suggesting its potential roles in Arabidopsis Cd tolerance. Consistent with this, a SR34b T-DNA insertion mutant (sr34b) was moderately sensitive to Cd, which had higher Cd{sup 2+} uptake rate and accumulated Cd in greater amounts than wild-type. This was due to the altered expression of iron-regulated transporter 1 (IRT1) gene in sr34b mutant. Under normal growth conditions, IRT1 mRNAs highly accumulated in sr34b mutant, which was a result of increased stability of IRT1 mRNA. Under Cd stress, however, sr34b mutant plants had a splicing defect in IRT1 gene, thus reducing the IRT1 mRNA accumulation. Despite of this, sr34b mutant plants still constitutively expressed IRT1 proteins under Cd stress, thereby resulting in Cd stress-sensitive phenotype. We therefore propose the essential roles of SR34b in posttranscriptional regulation of IRT1 expression and identify it as a regulator of Arabidopsis Cd tolerance.« less

Effect of short-term training on GLUT-4 mRNA and protein expression in human skeletal muscle.

PubMed

Kraniou, Giorgos N; Cameron-Smith, David; Hargreaves, Mark

2004-09-01

Six untrained, male subjects (23 +/- 1 years old, 84 +/- 5 kg, (O(2)peak)= 3.7 +/- 0.8 l min(-1)) exercised for 60 min at 75 +/- 1%(O(2)peak) on 7 consecutive days. Muscle samples were obtained before the start of cycle exercise training and 24 h after the first and seventh exercise sessions and analysed for citrate synthase activity, glycogen and glucose transporter 4 (GLUT-4) mRNA and protein expression. Exercise training increased (P < 0.05) citrate synthase by approximately 20% and muscle glycogen concentration by approximately 40%. GLUT-4 mRNA levels 24 h after the first and seventh exercise sessions were similar to those measured before the start of exercise training. In contrast, GLUT-4 protein expression was increased after 7 days of exercise training (12.4 +/- 1.5 versus 3.4 +/- 1.0 arbitray units (a.u.), P < 0.05) and although it tended to be higher 24 h after the first exercise session (6.0 +/- 3.0 versus 3.4 +/- 1.0 a.u.), this was not significantly different (P= 0.09). These results support the suggestion that the adaptive increase in skeletal muscle GLUT-4 protein expression with short-term exercise training arises from the repeated, transient increases in GLUT-gene transcription following each exercise bout leading to a gradual accumulation of GLUT-4 protein, despite GLUT-4 mRNA returning to basal levels between exercise stimuli.

Possible involvement of nuclear factor erythroid 2-related factor 2 in the gene expression of Cyp2b10 and Cyp2a5.

PubMed

Ashino, Takashi; Ohkubo-Morita, Haruyo; Yamamoto, Masayuki; Yoshida, Takemi; Numazawa, Satoshi

2014-01-01

Cytochrome P450 gene expression is altered by various chemical compounds. In this study, we used nuclear factor erythroid 2-related factor 2 (Nrf2)-deficient (Nrf2(-⧸-)) mice to investigate the involvement of Nrf2 in Cyp2b10 and Cyp2a5 gene expression. Phorone, an Nrf2 activator, strongly increased Cyp2b10 and Cyp2a5 mRNA as well as Nrf2 target genes, including NAD(P)H-quinone oxidoreductase-1 and heme oxygenase-1, in wild-type mouse livers 8 h after treatment. The phorone-induced mRNA levels in Nrf2(-⧸-) mouse livers were lower than that in wild-type mouse livers. Nrf2(-⧸-) mice showed attenuated Cyp2b10 and Cyp2a5 induction by phenobarbital, a classical Cyp2b inducer. These findings suggest that the Nrf2 pathway is involved in Cyp2b10 and Cyp2a5 gene expression.

Cloning of guinea pig IL-4: reduced IL-4 mRNA after vaccination or Mycobacterium tuberculosis infection.

PubMed

Jeevan, Amminikutty; Yoshimura, Teizo; Ly, Lan H; Dirisala, Vijaya R; McMurray, David N

2011-01-01

Interleukin-4 (IL-4), a pleiotropic cytokine produced by T-helper type 2 (Th2) cells, is involved in promoting humoral immune responses, allergic reactions and asthma. Previous studies suggested an important role for IL-4 in susceptibility to pulmonary tuberculosis; however, the role of IL-4 has not been studied in the guinea pig, a highly relevant model for this disease. In the present study, we cloned a cDNA for guinea pig IL-4 and examined, for the first time, mRNA expression by real-time RT-PCR in cultured guinea pig cells. High levels of IL-4 mRNA expression were detected in spleen T cells of naïve animals after in vitro stimulation with PMA plus ionomycin for 4-24 h. The expression of IL-4 mRNA was low in spleen and lymph node cells immunized with ovalbumin (OVA) plus Complete Freund's Adjuvant (CFA) in response to OVA (Th1), but significantly higher in the guinea pigs immunized with OVA plus alum (Th2). BCG vaccination reduced the expression of IL-4 mRNA in both spleen and lung digest cells compared to naïve guinea pigs, while levels of IFN-γ were similar in both groups. Furthermore, lung cells from Mycobacterium tuberculosis-infected guinea pigs stimulated in vitro with PPD or MPT64 showed low levels of IL-4 mRNA expression. Thus, BCG vaccination or M. tuberculosis infection modulates IL-4 mRNA expression in the guinea pig. Cloning of guinea pig IL-4 will allow us to address the role of IL-4 in vaccine-induced resistance to pulmonary TB in a highly relevant animal model. Copyright © 2010 Elsevier Ltd. All rights reserved.

Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses.

PubMed

Pardi, Norbert; Hogan, Michael J; Naradikian, Martin S; Parkhouse, Kaela; Cain, Derek W; Jones, Letitia; Moody, M Anthony; Verkerke, Hans P; Myles, Arpita; Willis, Elinor; LaBranche, Celia C; Montefiori, David C; Lobby, Jenna L; Saunders, Kevin O; Liao, Hua-Xin; Korber, Bette T; Sutherland, Laura L; Scearce, Richard M; Hraber, Peter T; Tombácz, István; Muramatsu, Hiromi; Ni, Houping; Balikov, Daniel A; Li, Charles; Mui, Barbara L; Tam, Ying K; Krammer, Florian; Karikó, Katalin; Polacino, Patricia; Eisenlohr, Laurence C; Madden, Thomas D; Hope, Michael J; Lewis, Mark G; Lee, Kelly K; Hu, Shiu-Lok; Hensley, Scott E; Cancro, Michael P; Haynes, Barton F; Weissman, Drew

2018-06-04

T follicular helper (Tfh) cells are required to develop germinal center (GC) responses and drive immunoglobulin class switch, affinity maturation, and long-term B cell memory. In this study, we characterize a recently developed vaccine platform, nucleoside-modified, purified mRNA encapsulated in lipid nanoparticles (mRNA-LNPs), that induces high levels of Tfh and GC B cells. Intradermal vaccination with nucleoside-modified mRNA-LNPs encoding various viral surface antigens elicited polyfunctional, antigen-specific, CD4 + T cell responses and potent neutralizing antibody responses in mice and nonhuman primates. Importantly, the strong antigen-specific Tfh cell response and high numbers of GC B cells and plasma cells were associated with long-lived and high-affinity neutralizing antibodies and durable protection. Comparative studies demonstrated that nucleoside-modified mRNA-LNP vaccines outperformed adjuvanted protein and inactivated virus vaccines and pathogen infection. The incorporation of noninflammatory, modified nucleosides in the mRNA is required for the production of large amounts of antigen and for robust immune responses. © 2018 Pardi et al.

In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity.

PubMed

Mazzari, Andre L D A; Milton, Flora; Frangos, Samantha; Carvalho, Ana C B; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M

2016-01-01

Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p < 0.05), this being correlated with an antagonist effect upon hPXR (EC50 = 0.38 mg/mL). Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p < 0.001), Lippia sidoides (-12%, p < 0.05) and Cordia verbenacea (-47%, p < 0.001). The latter plant extract was able to decrease GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum.

In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity

PubMed Central

Mazzari, Andre L. D. A.; Milton, Flora; Frangos, Samantha; Carvalho, Ana C. B.; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M.

2016-01-01

Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p < 0.05), this being correlated with an antagonist effect upon hPXR (EC50 = 0.38 mg/mL). Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p < 0.001), Lippia sidoides (-12%, p < 0.05) and Cordia verbenacea (-47%, p < 0.001). The latter plant extract was able to decrease GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum. PMID:27594838

Population density approach for discrete mRNA distributions in generalized switching models for stochastic gene expression.

PubMed

Stinchcombe, Adam R; Peskin, Charles S; Tranchina, Daniel

2012-06-01

We present a generalization of a population density approach for modeling and analysis of stochastic gene expression. In the model, the gene of interest fluctuates stochastically between an inactive state, in which transcription cannot occur, and an active state, in which discrete transcription events occur; and the individual mRNA molecules are degraded stochastically in an independent manner. This sort of model in simplest form with exponential dwell times has been used to explain experimental estimates of the discrete distribution of random mRNA copy number. In our generalization, the random dwell times in the inactive and active states, T_{0} and T_{1}, respectively, are independent random variables drawn from any specified distributions. Consequently, the probability per unit time of switching out of a state depends on the time since entering that state. Our method exploits a connection between the fully discrete random process and a related continuous process. We present numerical methods for computing steady-state mRNA distributions and an analytical derivation of the mRNA autocovariance function. We find that empirical estimates of the steady-state mRNA probability mass function from Monte Carlo simulations of laboratory data do not allow one to distinguish between underlying models with exponential and nonexponential dwell times in some relevant parameter regimes. However, in these parameter regimes and where the autocovariance function has negative lobes, the autocovariance function disambiguates the two types of models. Our results strongly suggest that temporal data beyond the autocovariance function is required in general to characterize gene switching.

Coexpression of the KCNA3B gene product with Kv1.5 leads to a novel A-type potassium channel.

PubMed

Leicher, T; Bähring, R; Isbrandt, D; Pongs, O

1998-12-25

Shaker-related voltage-gated potassium (Kv) channels may be heterooligomers consisting of membrane-integral alpha-subunits associated with auxiliary cytoplasmic beta-subunits. In this study we have cloned the human Kvbeta3.1 subunit and the corresponding KCNA3B gene. Identification of sequence-tagged sites in the gene mapped KCNA3B to band p13.1 of human chromosome 17. Comparison of the KCNA1B, KCNA2B, and KCNA3B gene structures showed that the three Kvbeta genes have very disparate lengths varying from >/=350 kb (KCNA1B) to approximately 7 kb (KCNA3B). Yet, the exon patterns of the three genes, which code for the seven known mammalian Kvbeta subunits, are very similar. The Kvbeta1 and Kvbeta2 splice variants are generated by alternative use of 5'-exons. Mouse Kvbeta4, a potential splice variant of Kvbeta3, is a read-through product where the open reading frame starts within the sequence intervening between Kvbeta3 exons 7 and 8. The human KCNA3B sequence does not contain a mouse Kvbeta4-like open reading frame. Human Kvbeta3 mRNA is specifically expressed in the brain, where it is predominantly detected in the cerebellum. The heterologous coexpression of human Kv1.5 and Kvbeta3.1 subunits in Chinese hamster ovary cells yielded a novel Kv channel mediating very fast inactivating (A-type) outward currents upon depolarization. Thus, the expression of Kvbeta3.1 subunits potentially extends the possibilities to express diverse A-type Kv channels in the human brain.

Direct Reprogramming of Human Amniotic Fluid Stem Cells by OCT4 and Application in Repairing of Cerebral Ischemia Damage

PubMed Central

Qin, Mingde; Chen, Ruihua; Li, Hong; Liang, Hansi; Xue, Qun; Li, Fang; Chen, Ying; Zhang, Xueguang

2016-01-01

Amniotic fluid stem cells (AFSCs) are a type of fetal stem cell whose stemness encompasses both embryonic and adult stem cells, suggesting that they may be easily and efficiently reprogrammed into induced pluripotent stem cells (iPSCs). To further simplify the reprogramming process, the creation of AFSC-derived iPSCs using a single factor is desirable. Here we report the generation of one-factor human AFSC-iPSCs (AiPSCs) from human AFSCs by ectopic expression of the transcription factor OCT4. Just like human embryonic stem cells, AiPSCs exhibited similar epigenetic status, global gene expression profiles, teratoma formation and in vitro & in vivo pluripotency. Our results indicate that the OCT4 is necessary and sufficient to directly reprogram human AFSCs into pluripotent AiPSCs. Moreover, reflecting the similar memory characteristics of AFSCs and neural stem cells, we show that AiPSC membrane-derived vesicles (MVs) repair cerebral ischemia damage. We anticipate that the successful generation of one-factor AiPSCs will facilitate the creation of patient-specific pluripotent stem cells without the need for transgenic expression of oncogenes. Moreover, MVs from tissue-specific AiPSCs have potential in tissue repair, representing a novel application of iPSCs. PMID:27019637

m6A-Driver: Identifying Context-Specific mRNA m6A Methylation-Driven Gene Interaction Networks

PubMed Central

Zhang, Song-Yao; Zhang, Shao-Wu; Liu, Lian; Huang, Yufei

2016-01-01

As the most prevalent mammalian mRNA epigenetic modification, N6-methyladenosine (m6A) has been shown to possess important post-transcriptional regulatory functions. However, the regulatory mechanisms and functional circuits of m6A are still largely elusive. To help unveil the regulatory circuitry mediated by mRNA m6A methylation, we develop here m6A-Driver, an algorithm for predicting m6A-driven genes and associated networks, whose functional interactions are likely to be actively modulated by m6A methylation under a specific condition. Specifically, m6A-Driver integrates the PPI network and the predicted differential m6A methylation sites from methylated RNA immunoprecipitation sequencing (MeRIP-Seq) data using a Random Walk with Restart (RWR) algorithm and then builds a consensus m6A-driven network of m6A-driven genes. To evaluate the performance, we applied m6A-Driver to build the context-specific m6A-driven networks for 4 known m6A (de)methylases, i.e., FTO, METTL3, METTL14 and WTAP. Our results suggest that m6A-Driver can robustly and efficiently identify m6A-driven genes that are functionally more enriched and associated with higher degree of differential expression than differential m6A methylated genes. Pathway analysis of the constructed context-specific m6A-driven gene networks further revealed the regulatory circuitry underlying the dynamic interplays between the methyltransferases and demethylase at the epitranscriptomic layer of gene regulation. PMID:28027310

Targeting the MIR34C-5p-ATG4B-autophagy axis enhances the sensitivity of cervical cancer cells to pirarubicin.

PubMed

Wu, Yaran; Ni, Zhenhong; Yan, Xiaojing; Dai, Xufang; Hu, Changjiang; Zheng, Yingru; He, Fengtian; Lian, Jiqin

2016-07-02

Pirarubicin (THP) is a newer generation anthracycline anticancer drug. In the clinic, THP and THP-based combination therapies have been demonstrated to be effective against various tumors without severe side effects. However, previous clinical studies have shown that most patients with cervical cancer are not sensitive to THP treatment, and the associated mechanisms are not clear. Consistent with the clinical study, we confirmed that cervical cancer cells were resistant to THP in vitro and in vivo. Our data demonstrated that THP induced a protective macroautophagy/autophagy response in cervical cancer cells, and suppression of this autophagy dramatically enhanced the cytotoxicity of THP. By scanning the mRNA level change of autophagy-related genes, we found that the upregulation of ATG4B (autophagy-related 4B cysteine peptidase) plays an important role in THP-induced autophagy. Moreover, THP increased the mRNA level of ATG4B in cervical cancer cells by promoting mRNA stability without influencing its transcription. Furthermore, THP triggered a downregulation of MIR34C-5p, which was associated with the upregulation of ATG4B and autophagy induction. Overexpression of MIR34C-5p significantly decreased the level of ATG4B and attenuated autophagy, accompanied by enhanced cell death and apoptosis in THP-treated cervical cancer cells. These results for the first time reveal the presence of a MIR34C-5p-ATG4B-autophagy signaling axis in THP-treated cervical cancer cells in vitro and in vivo, and the axis, at least partially, accounts for the THP nonsensitivity in cervical cancer patients. This study may provide a new insight for improving the chemotherapeutic effect of THP, which may be beneficial to the further clinical application of THP in cervical cancer treatment.

Targeting the MIR34C-5p-ATG4B-autophagy axis enhances the sensitivity of cervical cancer cells to pirarubicin

PubMed Central

Wu, Yaran; Ni, Zhenhong; Yan, Xiaojing; Dai, Xufang; Hu, Changjiang; Zheng, Yingru; He, Fengtian; Lian, Jiqin

2016-01-01

ABSTRACT Pirarubicin (THP) is a newer generation anthracycline anticancer drug. In the clinic, THP and THP-based combination therapies have been demonstrated to be effective against various tumors without severe side effects. However, previous clinical studies have shown that most patients with cervical cancer are not sensitive to THP treatment, and the associated mechanisms are not clear. Consistent with the clinical study, we confirmed that cervical cancer cells were resistant to THP in vitro and in vivo. Our data demonstrated that THP induced a protective macroautophagy/autophagy response in cervical cancer cells, and suppression of this autophagy dramatically enhanced the cytotoxicity of THP. By scanning the mRNA level change of autophagy-related genes, we found that the upregulation of ATG4B (autophagy-related 4B cysteine peptidase) plays an important role in THP-induced autophagy. Moreover, THP increased the mRNA level of ATG4B in cervical cancer cells by promoting mRNA stability without influencing its transcription. Furthermore, THP triggered a downregulation of MIR34C-5p, which was associated with the upregulation of ATG4B and autophagy induction. Overexpression of MIR34C-5p significantly decreased the level of ATG4B and attenuated autophagy, accompanied by enhanced cell death and apoptosis in THP-treated cervical cancer cells. These results for the first time reveal the presence of a MIR34C-5p-ATG4B-autophagy signaling axis in THP-treated cervical cancer cells in vitro and in vivo, and the axis, at least partially, accounts for the THP nonsensitivity in cervical cancer patients. This study may provide a new insight for improving the chemotherapeutic effect of THP, which may be beneficial to the further clinical application of THP in cervical cancer treatment. PMID:27097054

Programmed cell death 4 protein (Pdcd4) and homeodomain-interacting protein kinase 2 (Hipk2) antagonistically control translation of Hipk2 mRNA.

PubMed

Ohnheiser, Johanna; Ferlemann, Eva; Haas, Astrid; Müller, Jan P; Werwein, Eugen; Fehler, Olesja; Biyanee, Abhiruchi; Klempnauer, Karl-Heinz

2015-07-01

The tumor suppressor protein programmed cell death 4 (Pdcd4) is a highly conserved RNA-binding protein that inhibits the translation of specific mRNAs. Here, we have identified the homeobox-interacting protein kinase-2 (Hipk2) mRNA as a novel translational target of Pdcd4. Unlike most other protein kinases Hipk2 is constitutively active after being synthesized by the ribosome and its expression and activity are thought to be mainly controlled by modulation of the half-life of the kinase. Our work provides the first evidence that Hipk2 expression is also controlled on the level of translation. We show that Hipk2 stimulates the translation of its own mRNA and that Pdcd4 suppresses the translation of Hipk2 mRNA by interfering with this auto-regulatory feedback mechanism. We also show that the translation of the related kinase Hipk1 is controlled by a similar feedback loop and that Hipk2 also stimulates the translation of Hipk1 mRNA. Taken together, our work describes a novel mechanism of translational suppression by Pdcd4 and shows for the first time that Hipk2 controls its own synthesis by an auto-regulatory feedback mechanism. Furthermore, the effect of Hipk2 on the translation of Hipk1 RNA suggests that Hipk2 and Pdcd4 can act in similar manner to control the translation of other mRNAs. Copyright © 2015 Elsevier B.V. All rights reserved.

Aging alters mRNA expression of amyloid transporter genes at the blood-brain barrier.

PubMed

Osgood, Doreen; Miller, Miles C; Messier, Arthur A; Gonzalez, Liliana; Silverberg, Gerald D

2017-09-01

Decreased clearance of potentially toxic metabolites, due to aging changes, likely plays a significant role in the accumulation of amyloid-beta (Aβ) peptides and other macromolecules in the brain of the elderly and in the patients with Alzheimer's disease (AD). Aging is the single most important risk factor for AD development. Aβ transport receptor proteins expressed at the blood-brain barrier are significantly altered with age: the efflux transporters lipoprotein receptor-related protein 1 and P-glycoprotein are reduced, whereas the influx transporter receptor for advanced glycation end products is increased. These receptors play an important role in maintaining brain biochemical homeostasis. We now report that, in a rat model of aging, gene transcription is altered in aging, as measured by Aβ receptor gene messenger RNA (mRNA) at 3, 6, 9, 12, 15, 20, 30, and 36 months. Gene mRNA expression from isolated cerebral microvessels was measured by quantitative polymerase chain reaction. Lipoprotein receptor-related protein 1 and P-glycoprotein mRNA were significantly reduced in aging, and receptor for advanced glycation end products was increased, in parallel with the changes seen in receptor protein expression. Transcriptional changes appear to play a role in aging alterations in blood-brain barrier receptor expression and Aβ accumulation. Copyright © 2017 Elsevier Inc. All rights reserved.

An exonic missense mutation c.28G>A is associated with weak B blood group by affecting RNA splicing of the ABO gene.

PubMed

Cai, Xiaohong; Qian, Chengrui; Wu, Wenman; Lei, Hang; Ding, Qiulan; Zou, Wei; Xiang, Dong; Wang, Xuefeng

2017-09-01

The amino acid substitutions caused by ABO gene mutations are usually predicted to impact glycosyltransferase's function or its biosynthesis. Here we report an ABO exonic missense mutation that affects B-antigen expression by decreasing the mRNA level of the ABO gene rather than the amino acid change. Serologic studies including plasma total GTB transfer capacity were performed. The exon sequences of the ABO gene were analyzed by Sanger sequencing. B 310 cDNA with c.28G>A (p.G10R) mutation was expressed in HeLa cells and total GTB transfer capacity in cell supernatant was measured. Flow cytometry was performed on these HeLa cells after transfection, and agglutination of Hela-B weak cells was also examined. The mRNA of the ABO gene was analyzed by direct sequencing and real-time reverse transcriptase-polymerase chain reaction. A minigene construct was prepared to evaluate the potential of splicing. While plasma total GTB transfer capacity was undetectable in this B 3 -like individual, the relative percentage of antigen-expressing cells and mean fluorescence index of the B weak red blood cells (RBCs) were 19 and 14% of normal B RBCs, respectively. There was no significant difference of total GTB transfer capacity in cell supernatant and B-antigen expression on cell surfaces between HeLa cells transfected with B 310 cDNA and B cDNA. The mRNA expression level of B 310 in peripheral whole blood was significantly reduced. The amount of splicing is significantly lower in c.28G>A construct compared to that in wild-type construct after transfection in K562 cells. ABO c.28G>A mutation may cause B 3 -like subgroup by affecting RNA splicing of the ABO gene. © 2017 AABB.

[Expression and clinical significance of KNSL4 in breast cancer].

PubMed

Feng, Yu-Mei; Wan, Yan-Fang; Li, Xiao-Qing; Cao, Xu-Chen; Li, Xi

2006-06-01

Previous screening of breast cancer metastasis-related genes found that the mRNA level of kinesin-like 4 (KNSL4) gene is down-regulated in metastatic lymph nodes as compared with the paired primary breast cancer. This study was to clarify the correlations of KNSL4 mRNA expression to metastasis and prognosis of breast cancer, and explore the correlation of KNSL4 expression to c-erbB-2 expression to explore potential mechanisms of promoting metastasis by KNSL4. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify the mRNA level of KNSL4 in 108 specimens of primary breast cancer. The correlations of KNSL4 mRNA level to metastasis and prognosis of the 108 cases were analyzed. Immunohistochemistry was used to assess c-erbB-2 protien expression in 76 out of the 108 cases, and the correlation of KNSL4 expression to c-erbB-2 expression was analyzed. The mRNA level of KNSL4 was significantly lower in the cases at stages iii-iv than in the cases at stages iii-iv (P<0.001), significantly lower in the cases with more than 3 metastatic lymph nodes than in the cases with 0-3 metastatic positive lymph nodes (P<0.01), slightly lower in the cases with negative estrogen receptor or prognesterone receptor than in the cases with positive receptors (P>0.05), lower in the 6 cases with distant metastasis than in the rest cases without distant metastasis within 24 month follow up, lower in the 3 cases with bilateral breast cancer than in other cases with unilateral breast cancer, and significantly lower in c-erbB-2-positive group than in c-erB-2-negative group (P<0.01). The down-regulation of KNSL4 mRNA level is correlated to prognosis of primary breast cancer. It may enhance metastatic ability of breast cancer cells through promoting c-erbB-2 transcription and translation.

Molecular Cloning and Characterization of the Human ErbB4 Gene: Identification of Novel Splice Isoforms in the Developing and Adult Brain

PubMed Central

Tan, Wei; Dean, Michael; Law, Amanda J.

2010-01-01

ErbB4 is a growth factor receptor tyrosine kinase essential for neurodevelopment. Genetic variation in ErbB4 is associated with schizophrenia and risk-associated polymorphisms predict overexpression of ErbB4 CYT-1 isoforms in the brain in the disorder. The molecular mechanism of association is unclear because the polymorphisms flank exon 3 of the gene and reside 700 kb distal to the CYT-1 defining exon. We hypothesized that the polymorphisms are indirectly associated with ErbB4 CYT-1 via splicing of exon 3 on the CYT-1 background. We report via cloning and sequencing of adult and fetal human brain cDNA libraries the identification of novel splice isoforms of ErbB4, whereby exon 3 is skipped (del.3). ErbB4 del.3 transcripts exist as CYT-2 isoforms and are predicted to produce truncated proteins. Furthermore, our data refine the structure of the human ErbB4 gene, clarify that juxtamembrane (JM) splice variants of ErbB4, JM-a and JM-b respectively, are characterized by the replacement of a 75 nucleotide (nt) sequence with a 45-nt insertion, and demonstrate that there are four alternative exons in the gene. Our analyses reveal that novel splice variants of ErbB4 exist in the developing and adult human brain and, given the failure to identify ErbB4 del.3 CYT-1 transcripts, suggest that the association of risk polymorphisms in the ErbB4 gene with CYT-1 transcript levels is not mediated via an exon 3 splicing event. PMID:20886074

Intergenic mRNA molecules resulting from trans-splicing.

PubMed

Finta, Csaba; Zaphiropoulos, Peter G

2002-02-22

Accumulated recent evidence is indicating that alternative splicing represents a generalized process that increases the complexity of human gene expression. Here we show that mRNA production may not necessarily be limited to single genes, as human liver also has the potential to produce a variety of hybrid cytochrome P450 3A mRNA molecules. The four known cytochrome P450 3A genes in humans, CYP3A4, CYP3A5, CYP3A7, and CYP3A43, share a high degree of similarity, consist of 13 exons with conserved exon-intron boundaries, and form a cluster on chromosome 7. The chimeric CYP3A mRNA molecules described herein are characterized by CYP3A43 exon 1 joined at canonical splice sites to distinct sets of CYP3A4 or CYP3A5 exons. Because the CYP3A43 gene is in a head-to-head orientation with the CYP3A4 and CYP3A5 genes, bypassing transcriptional termination can not account for the formation of hybrid CYP3A mRNAs. Thus, the mechanism generating these molecules has to be an RNA processing event that joins exons of independent pre-mRNA molecules, i.e. trans-splicing. Using quantitative real-time polymerase chain reaction, the ratio of one CYP3A43/3A4 intergenic combination was estimated to be approximately 0.15% that of the CYP3A43 mRNAs. Moreover, trans-splicing has been found not to interfere with polyadenylation. Heterologous expression of the chimeric species composed of CYP3A43 exon 1 joined to exons 2-13 of CYP3A4 revealed catalytic activity toward testosterone.

Vitamin B1 diversity and characterization of biosynthesis genes in cassava.

PubMed

Mangel, Nathalie; Fudge, Jared B; Fitzpatrick, Teresa B; Gruissem, Wilhelm; Vanderschuren, Hervé

2017-06-15

Vitamin B1, which consists of the vitamers thiamin and its phosphorylated derivatives, is an essential micronutrient for all living organisms because it is required as a metabolic cofactor in several enzymatic reactions. Genetic diversity of vitamin B1 biosynthesis and accumulation has not been investigated in major crop species other than rice and potato. We analyzed cassava germplasm for accumulation of B1 vitamers. Vitamin B1 content in leaves and roots of 41 cassava accessions showed significant variation between accessions. HPLC analyses of B1 vitamers revealed distinct profiles in cassava leaves and storage roots, with nearly equal relative levels of thiamin pyrophosphate and thiamin monophosphate in leaves, but mostly thiamin pyrophosphate in storage roots. Unusually, the cassava genome has two genes encoding the 4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate synthase, THIC (MeTHIC1 and MeTHIC2), both of which carry a riboswitch in the 3'-UTR, as well as the adenylated thiazole synthase, THI1 (MeTHI1a and MeTHI1b). The THIC and THI1 genes are expressed at very low levels in storage roots compared with the accumulation of vitamin B1, indicating only limited biosynthesis de novo therein. In leaves, vitamin B1 content is negatively correlated with THIC and THI1 expression levels, suggesting post-transcriptional regulation of THIC by the riboswitch present in the 3'-UTR of the THIC mRNA and regulation of THI1 by promoter activity or alternative post-transcriptional mechanisms. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Imaging Transcriptional Regulation of Eukaryotic mRNA Genes: Advances and Outlook.

PubMed

Yao, Jie

2017-01-06

Regulation of eukaryotic transcription in vivo occurs at distinct stages. Previous research has identified many active or repressive transcription factors (TFs) and core transcription components and studied their functions in vitro and in vivo. Nonetheless, how individual TFs act in concert to regulate mRNA gene expression in a single cell remains poorly understood. Direct observation of TF assembly and disassembly and various biochemical reactions during transcription of a single-copy gene in vivo is the ideal approach to study this problem. Research in this area requires developing novel techniques for single-cell transcription imaging and integrating imaging studies into understanding the molecular biology of transcription. In the past decade, advanced cell imaging has enabled unprecedented capabilities to visualize individual TF molecules, to track single transcription sites, and to detect individual mRNA in fixed and living cells. These studies have raised several novel insights on transcriptional regulation such as the "hit-and-run" model and transcription bursting that could not be obtained by in vitro biochemistry analysis. At this point, the key question is how to achieve deeper understandings or discover novel mechanisms of eukaryotic transcriptional regulation by imaging transcription in single cells. Meanwhile, further technical advancements are likely required for visualizing distinct kinetic steps of transcription on a single-copy gene in vivo. This review article summarizes recent progress in the field and describes the challenges and opportunities ahead. Copyright © 2016 Elsevier Ltd. All rights reserved.

Regulation of early mRNA synthesis after bacteriophage T4 infection of Escherichia coli.

PubMed Central

Linder, C H; Fast, R

1975-01-01

Regulation of T4-specific mRNA synthesis was studied during leucine starvation of a leucine-requiring stringent Escherichia coli B strain. This was done by imposing starvation prior to T4 infection and then letting RNA synthesis proceed for different time periods. Rifampin or streptolydigin was added to stop further RNA synthesis, and protein synthesis was restored by addition of leucine. Samples were withdrawn at different times, and the enzyme-forming capacities found that, during conditions which elicit the stringent response in uninfected bacteria, immediate early mRNA is not stringently regulated. This conclusion contradicts the earlier conclusion of others, obtained by measuring incorporation of radioactive uracil; this is explained by the observation of Edlin and Neuhard (1967), confirmed and extended by us to the T4-infected cell, that the incorporation of uracil into RNA of a stringent strain is virtually blocked by amino acid starvation, whereas that of adenine continues at 30 to 50% of the rate seen in the presence of the required amino acid. PMID:1099229

Association of mRNA expression of iron metabolism-associated genes and progression of non-alcoholic steatohepatitis in rats.

PubMed

Higuchi, Teruhisa; Moriyama, Mitsuhiko; Fukushima, Akiko; Matsumura, Hiroshi; Matsuoka, Shunichi; Kanda, Tatsuo; Sugitani, Masahiko; Tsunemi, Akiko; Ueno, Takahiro; Fukuda, Noboru

2018-05-25

Excess iron is associated with non-alcoholic steatohepatitis (NASH). mRNA expression of duodenal cytochrome b, divalent metal transporter 1, ferroportin 1, hepcidin, hephaestin and transferrin receptor 1 in liver were higher in high fat, high cholesterol-containing diet (HFCD) group than in normal diet (ND) group. mRNA levels of divalent metal transporter 1 and transferrin receptor 1, which stimulate iron absorption and excretion, were enhanced in small intestine. Epithelial mucosa of small intestine in HFCD group was characterized by plasma cell and eosinophil infiltration and increased vacuoles. Iron absorption was enhanced in this NASH model in the context of chronic inflammation of small intestinal epithelial cells, consequences of intestinal epithelial cell impairment caused by HFCD. Iron is transported to hepatocytes via portal blood, and abnormalities in iron absorption and excretion occur in small intestine from changes in iron transporter expression, which also occurs in NASH liver. Knockdown of hepcidin antimicrobial peptide led to enhanced heavy chain of ferritin expression in human hepatocytes, indicating association between hepcidin production and iron storage in hepatocytes. Iron-related transporters in liver and lower/upper portions of small intestine play critical roles in NASH development. Expression of iron metabolism-related genes in liver and small intestine was analyzed in stroke-prone spontaneously hypertensive rats (SHR-SP), which develop NASH. Five-week-old SHR-SP fed ND or HFCD were examined. mRNA and protein levels of iron metabolism-related genes in liver and small intestine from 12- and 19-week-old rats were evaluated by real-time RT-PCR and immunohistochemistry or Western blot.

Follicular progesterone concentrations and messenger RNA expression of MATER and OCT-4 in immature bovine oocytes as predictors of developmental competence.

PubMed

Urrego, R; Herrera-Puerta, E; Chavarria, N A; Camargo, O; Wrenzycki, C; Rodriguez-Osorio, N

2015-04-15

The ability of bovine embryos to develop to the blastocyst stage and to implant and generate healthy offspring depends greatly on the competence of the oocyte. Oocyte competence is attributed to its close communication with the follicular environment and to its capacity to synthesize and store substantial amounts of messenger RNA. Higher developmental competence of bovine oocytes has been associated with both the expression of a cohort of developmental genes and the concentration of sex steroids in the follicular fluid. The aim of this study was to identify differences in the expression of FST in cumulus cells and OCT-4 and MATER in oocytes and the influence of the follicular progesterone and follicular estrogen concentration on the competence of bovine oocytes retrieved 30 minutes or 4 hours after slaughter. Cumulus-oocyte complexes (COCs) were left in postmortem ovaries for 30 minutes (group I) or 4 hours (group II) at 30 °C. Aspirated oocytes were then subjected to IVM, IVF, and IVC or were evaluated for MATER and OCT-4 messenger RNA abundance by quantitative real-time polymerase chain reaction. Total RNA was isolated from pools of 100 oocytes for each experimental replicate. Progesterone and estradiol concentration in follicular fluid was evaluated by immunoassay using an IMMULITE 2000 analyzer. Three repeats of in vitro embryo production were performed with a total of 455 (group I) and 470 (group II) COCs. There were no significant differences between the cleavage rates (72 hours postinsemination [hpi]) between both groups (63.5% vs. 69.1%). However, blastocyst (168 hpi) and hatching (216 hpi) rates were higher (P < 0.05) in group II compared with those of group I (21.3% vs. 30.7% and 27.6% vs. 51.5%, respectively). Group II oocytes exhibited the highest MATER and OCT-4 abundance (P < 0.05). Follicular estradiol concentration was not different between both the groups, whereas the progesterone concentration was lower (P ≤ 0.05) in group II follicles. These

The Role of CYP3A4 mRNA Transcript with Shortened 3′-Untranslated Region in Hepatocyte Differentiation, Liver Development, and Response to Drug InductionS⃞

PubMed Central

Li, Dan; Gaedigk, Roger; Hart, Steven N.; Leeder, J. Steven

2012-01-01

Cytochrome P450 3A4 (CYP3A4) metabolizes more than 50% of prescribed drugs. The expression of CYP3A4 changes during liver development and may be affected by the administration of some drugs. Alternative mRNA transcripts occur in more than 90% of human genes and are frequently observed in cells responding to developmental and environmental signals. Different mRNA transcripts may encode functionally distinct proteins or contribute to variability of mRNA stability or protein translation efficiency. The purpose of this study was to examine expression of alternative CYP3A4 mRNA transcripts in hepatocytes in response to developmental signals and drugs. cDNA cloning and RNA sequencing (RNA-Seq) were used to identify CYP3A4 mRNA transcripts. Three transcripts were found in HepaRG cells and liver tissues: one represented a canonical mRNA with full-length 3′-untranslated region (UTR), one had a shorter 3′-UTR, and one contained partial intron-6 retention. The alternative mRNA transcripts were validated by either rapid amplification of cDNA 3′-end or endpoint polymerase chain reaction (PCR). Quantification of the transcripts by RNA-Seq and real time quantitative PCR revealed that the CYP3A4 transcript with shorter 3′-UTR was preferentially expressed in developed livers, differentiated hepatocytes, and in rifampicin- and phenobarbital-induced hepatocytes. The CYP3A4 transcript with shorter 3′-UTR was more stable and produced more protein compared with the CYP3A4 transcript with canonical 3′-UTR. We conclude that the 3′-end processing of CYP3A4 contributes to the quantitative regulation of CYP3A4 gene expression through alternative polyadenylation, which may serve as a regulatory mechanism explaining changes of CYP3A4 expression and activity during hepatocyte differentiation and liver development and in response to drug induction. PMID:21998292

Human placental lactogen mRNA and its structural genes during pregnancy: quantitation with a complementary DNA.

PubMed Central

McWilliams, D; Callahan, R C; Boime, I

1977-01-01

A complementary DNA (cDNA) strand was transcribed from human placental lactogen (hPL) mRNA. Based on alkaline sucrose gradient centrifugation, the size of the cDNA was about 8 S, which would represent at least 80% of the hPL mRNA. Previously we showed that four to five times more hPL was synthesized in cell-free extracts derived from term as compared to first trimester placentas. Hybridization of the cDNA with RNA derived from placental tissue revealed that there was about four times more hPL mRNA sequences in total RNA from term placenta than in a comparable quantity of total first trimester RNA. Only background hybridization was observed when the cDNA was incubated with RNA prepared from human kidney. To test if this differential accumulation of hPL mRNA was the result of an amplification of hPL genes, we hybridized the labeled cDNA with cellular DNA from first trimester and term placentas and with DNA isolated from human brain. In all cases, the amount of hPL sequences was approximately two copies per haploid genome. Thus, the enhanced synthesis of hPL mRNA appears to result from a transcriptional activation rather than an amplification of the hPL gene. The increase likely reflects placental differentiation in which the proportion of syncytial trophoblast increases at term. Images PMID:66681

A spontaneous tRNA suppressor of a mutation in the Chlamydomonas reinhardtii nuclear MCD1 gene required for stability of the chloroplast petD mRNA

PubMed Central

Murakami, Shinya; Kuehnle, Katrin; Stern, David B.

2005-01-01

Numerous nuclear gene products are required for the correct expression of organellar genes. One such gene in the green alga Chlamydomonas reinhardtii is MCD1, whose product is required for stability of the chloroplast-encoded petD mRNA. In mcd1 mutants, which are non-photosynthetic, petD mRNA is degraded by a 5′–3′ exonuclease activity, resulting in a failure to synthesize its product, subunit IV of the cytochrome b 6/f complex. Here, we report the sequence of the wild-type MCD1 gene, which encodes a large and novel putative protein. Analysis of three mutant alleles showed that two harbored large deletions, but that one allele, mcd1-2, had a single base change resulting in a nonsense codon near the N-terminus. This same mutant allele can be suppressed by a second-site mutation in the nuclear MCD2 gene, whereas mcd2-1 cannot suppress the deletion in mcd1-1 (Esposito,D. Higgs,D.C. Drager,R.G. Stern, D.B. and Girard-Bascou,J. (2001) Curr. Genet., 39, 40–48). We report the cloning of mcd2-1, and show that the mutation lies in a tRNASer(CGA), which has been modified to translate the nonsense codon in mcd1-2. We discuss how the existence of a large tRNASer gene family may permit this suppression without pleiotropic consequences. PMID:15947135

Multiple PAR and E4BP4 bZIP transcription factors in zebrafish: diverse spatial and temporal expression patterns.

PubMed

Ben-Moshe, Zohar; Vatine, Gad; Alon, Shahar; Tovin, Adi; Mracek, Philipp; Foulkes, Nicholas S; Gothilf, Yoav

2010-09-01

Circadian rhythms of physiology and behavior are generated by an autonomous circadian oscillator that is synchronized daily with the environment, mainly by light input. The PAR subfamily of transcriptional activators and the related E4BP4 repressor belonging to the basic leucine zipper (bZIP) family are clock-controlled genes that are suggested to mediate downstream circadian clock processes and to feedback onto the core oscillator. Here, the authors report the characterization of these genes in the zebrafish, an increasingly important model in the field of chronobiology. Five novel PAR and six novel e4bp4 zebrafish homolog genes were identified using bioinformatic tools and their coding sequences were cloned. Based on their evolutionary relationships, these genes were annotated as ztef2, zhlf1 and zhlf2, zdbp1 and zdbp2, and ze4bp4-1 to -6. The spatial and temporal mRNA expression pattern of each of these factors was characterized in zebrafish embryos in the context of a functional circadian clock and regulation by light. Nine of the factors exhibited augmented and rhythmic expression in the pineal gland, a central clock organ in zebrafish. Moreover, these genes were found to be regulated, to variable extents, by the circadian clock and/or by light. Differential expression patterns of multiple paralogs in zebrafish suggest multiple roles for these factors within the vertebrate circadian clock. This study, in the genetically accessible zebrafish model, lays the foundation for further research regarding the involvement and specific roles of PAR and E4BP4 transcription factors in the vertebrate circadian clock mechanism.

Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming.

PubMed

Poon, Ming-Wai; He, Jia; Fang, Xiaowei; Zhang, Zhao; Wang, Weixin; Wang, Junwen; Qiu, Fangfang; Tse, Hung-Fat; Li, Wei; Liu, Zuguo; Lian, Qizhou

2015-01-01

A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs) generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs) exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming) and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs). Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2) reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%). Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells) was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs) cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs). This cell type may also have advantages in retinal pigmented epithelial differentiation.

Expression of p16(INK4A) gene in human pituitary tumours.

PubMed